Heteroclitic analogs and related methods

ABSTRACT

Heteroclitic analogs of Class I epitopes are prepared by providing conservative or semi-conservative amino acid substitutions at positions 3 and/or 5 and/or 7 of these epitopes. The analogs are useful in eliciting immune responses with respect to the corresponding wildtype epitopes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of InternationalApplication No. PCT/US00/31856, filed Nov. 20, 2000, which publishedunder PCT article 21(2) in English, and which claims the benefit of U.S.Provisional Patent Application No. 60/166,529, filed 18 Nov. 1999, andU.S. Provisional Patent Application No. 60/239,008, filed 6 Oct. 2000;each of said applications is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to methods for generating heteroclitic analogs ofan original peptide which have increased stimulatory capacity for agiven T cell.

Several studies suggest the cytotoxic T lymphocytes (CTLs) play acentral role in the eradication of infectious disease and cancer by theimmune system (Byrne, et al., J. Immunol. 51:682 (1984), McMichael, etal., N. England J. Med., 309:13 (1983)). Since CTLs are stimulated bypeptides comprising epitopes, considerable effort is ongoing indeveloping epitope-based vaccines that stimulate CTL responses. Oneclass of epitopes, designated heteroclitic analogs; provides benefit asvaccine components since these analogs induce T cell responses strongerthan those induced by the native epitope. Heteroclitic analogs aredefined as peptides having increased stimulatory capacity or potency fora specific T cell, as measured by increased responses to a given dose,or by a requirement of lesser amounts to achieve the same response.

The advantages associated with using heteroclitic analogs in clinicalapplications are as follows. First, heteroclitic analogs have theability to break/overcome tolerance by reversing a state of T cellanergy, activating non-tolerized cross-reactive clones of T cells, or bymediating “immune deviation,” i.e., the type of CTL produced, such asTh1 or Th2. Recent studies indicate that heteroclitic analogs areimmunogenic (Zaremba, et al., Cancer Research, 57:4570 (1997);Rivoltoni, et al., Cancer Research, 59:301 (1999); Selby, et al., 162(2):669 (1999)) in that they are capable of inducing CTLs that recognizeendogenously processed epitope. This is confirmed by studies indifferent immunological systems (Zugel, et al., J. Immunol., 161:1705(1998), Wang, et al., J. Exp. Med., 190:983 (1999), Men, et al., J.Immunol., 162:3566, (1999)). For example, studies by Zugel et al.(Zugel, et al, supra) have shown that T cell tolerance to animmunodominant T cell epitope in adult mice can be overcome byimmunization with heteroclitic cross-reactive peptide analogs of thatpeptide.

This is particularly significant in the field of cancer vaccines, wheremost of the CTL epitopes are derived from self antigens. Due to the factthat cancer related antigens are often self-antigens there is acorresponding phenomenon that there may be preexisting tolerance tothese antigens, whereby generation of a T cell response to such epitopesis a challenge. Breaking of tolerance by heteroclitic analogs has beenshown in a recent study in a murine Class II system (Wang, et al., J.Exp. Med. 190:983 (1999)). In this study, the mechanism involved inbreaking of tolerance was the stimulation of nontolerized, low affinityclones, rather than reversal of anergy. The heteroclicity demonstratedherein is associated with the induction of high avidity CTL, thisrepresents an important difference.

Second, peptide analogs have been demonstrated to modulate cytokineproduction from T cells (Pfeiffer, et al., J. Exp. Med., 181:1569(1995), Tao, et al., J. Immunol., 158:4237 (1997), Salazar, et al., Int.J. Cancer 85 (6):829-38 (2000), Nicholson, et al., Int. Immunol. 12(2):205-13 (2000)). The immune deviation induced by such analogs hasimplications in several disease states, where generation of a specificsubset of Th cell responses correlate with tumor regression (Zitvogel,et al., J. Exp. Med., 183:87 (1996), Celluzzi, et al., J. Exp. Med.183:283 (1996)) or affected the clinical outcome of autoimmune orinfectious disease (Romagnani, et al., Annu. Rev. Immunol., 12:227-57(1994)). Thus, immunization with heteroclitic analogs offers thecapacity to modulate cytokine production by induction of specificsubsets of effector T cells, thereby altering the course of disease.

Third, heteroclitic analogs offer an advantage in drug development sincesignificantly smaller amounts of peptide are needed for treatment doses,due to their strong biological potency. This feature overcomes certainmanufacturing and toxicity concerns. In this regard, it has been shownthat a heteroclitic analog of a MART-1 peptide (Rivoltini, et al.,Cancer Research 59:301 (1999)), which generated antigen specific T cellsin melanoma patients, was active at much lower concentrations than thenative epitope. Similar results were reported by Schlom and colleagues(Zaremba, et al., Cancer Research 57:4570 (1997)) regarding heterocliticanalog of the CEA derived CAP1 epitope. However, a side-by-sideprecursor frequency analysis or a TCR avidity analysis against wildtypepeptide was not performed.

Accordingly, because of their biological relevance, it would beextremely useful to predict amino acid substitutions that renderheteroclitic activity to a given epitope. However, prior to the presentdisclosure there has been no easy method for predicting suchsubstitutions. Indeed, in previous studies (Selby, et al., J. Immunol,162 (2):669 (1999), Skipper, et al., J. Exp. Med. 183:527 (1996)),heteroclitic epitopes were fortuitously identified by eluting naturallyoccurring mutant peptides from melanoma cells, or by systematicallyscreening a large number of analogs consisting of substitutions atalmost every position in the epitope (Zaremba, et al, Cancer Research,57:4570 (1997), Loftus, et al., Cancer Research 58:2433 (1998), Blake,et al., J. Exp. Med. 18:121 (1996)). Alternatively, heteroclitic analogswere identified by screening random combinatorial peptide librarieswhich also has required the arduous synthesis and screening of largenumbers of peptides (Pinilla, et al., Current Opinion in Immunology11:193-202 (1999)). Genetic approaches, such as screening of DNAexpression libraries, have provided another method for generating CTLepitopes and analogs (Boon, et al., Annu. Rev. Immunol. 12:337-65(1994), Gavin, et al., Eur. J. Immunol. 24 (9):2124-33 (1994)). However,this approach may be problematic given the potentially small quantitiesand complexity of epitopes generated.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods to prepare peptides containing epitopeswhich have enhanced ability to effect an immune response with respect tocorresponding analogous wildtype epitopes. The resulting “heterocliticanalogs” are useful in immunological compositions for treatment of viraldiseases, cancer, and other conditions which are characterized bydisplayed antigens on target cells.

Thus, in one aspect, the invention is directed to a method to enhancethe immunogenicity of a peptide containing an epitope, the methodcomprising i) providing a peptide comprising a first Class I epitopewherein said epitope consists essentially of an amino acid sequencehaving an N-terminus and a C-terminus and at least one primary anchorresidue, wherein amino acid residues of the epitope are numberedconsecutively and the primary anchor residue nearest the N-terminus ofthe epitope is at position 2 or position 3; and ii) introducing one ormore conservative or semi-conservative substitution between theN-terminus and the C-terminus of the epitope at position 3 and/or 5and/or 7 which position does not contain a primary anchor residue,thereby constructing a peptide comprising a second Class I epitope whichexhibits enhanced immunogenicity compared to the first Class I epitope.

In another aspect, in the case of B7 superfamily epitopes, the inventionis directed to a method to enhance the immunogenicity of a peptidecontaining a B7 superfamily epitope, the method comprising i) providinga peptide comprising a first Class I epitope which is a B7 superfamilyepitope wherein said epitope consists essentially of an amino acidsequence having an N-terminus and a C-terminus and at least one primaryanchor residue, wherein amino acid residues of the epitope are numberedconsecutively and the primary anchor residue nearest the N-terminus ofthe epitope is at position 2; and ii) introducing one or moreconservative, semi-conservative, or non-conservative substitutionbetween the N-terminus and the C-terminus of the epitope at position 3and/or 5 and/or 7, thereby constructing a peptide comprising a secondClass I epitope which is a B7 superfamily epitope which exhibitsenhanced immunogenicity compared to the first Class I epitope.

Thus, the invention relates to a method of producing a polypeptidecomprising an analog of a MHC class I epitope, wherein the analog hasenhanced immunogenicity compared to the epitope, comprising (a)identifying a MHC class I epitope comprising a formula (A), whereinformula (A) is Rn-R2-R3-R4-R5-R6-R7- . . . Rx, Rn is the N-terminalamino acid, Rx is the C-terminal amino acid, x=8-11 such that Rx can befrom the eighth to the eleventh amino acid residue from Rn, R2 or R3 andRx are primary anchor residues of a motif or supermotif, and (b)producing a polypeptide comprising an analog, said analog comprising aformula (B) identical to said formula (A) except one or moreconservative or semiconservative amino acid substitutions at R3 and/orR5 and/or R7, provided said one or more substitutions is not of aprimary anchor residues.

In some aspects, said analog comprises a formula (B) identical to saidformula (A) except that R3 is Met, provided R3 is not an anchor residueof said motif or supermotif.

In some aspects, said analog comprises a formula (B) identical to saidformula (A) except that R5 is Met.

In some aspects, said analog comprises a formula (B) identical to saidformula (A) except that R7 is Met.

In some aspects, R3 is Ile in formula (A), and said analog comprises aformula (B) identical to said formula (A) except that R3 is Met.

In some aspects, R3 is Lys in formula (A), and said analog comprises aformula (B) identical to said formula (A) except that R3 is His or Leu.

In some aspects, R5 is Val in formula (A), and said analog comprises aformula (B) identical to said formula (A) except that R5 is His.

In some aspects, R5 is Leu in formula (A), and said analog comprises aformula (B) identical to said formula (A) except that R5 is Ile.

In some aspects, R5 is Val in formula (A), and said analog comprises aformula (B) identical to said formula (A) except that R5 is Ile or Phe.

In some aspects, R7 is His in formula (A), and said analog comprises aformula (B) identical to said formula (A) except that R7 is Trp.

In some aspects, R7 is Ala in formula (A), and said analog comprises aformula (B) identical to said formula (A) except that R7 is Pro.

In some aspects, R7 is Tyr in formula (A), and said analog comprises aformula (B) identical to said formula (A) except that R7 is His or Met.

In other aspects, the invention relates to a method of producing apolypeptide comprising an analog of a MHC class I epitope, wherein theanalog has enhanced immunogenicity compared to the epitope, comprising(a) identifying a MHC class I epitope comprising a formula (A), whereinformula (A) is Rn-R2-R3-R4-R5-R6-R7- . . . Rx, Rn is the N-terminalamino acid, Rx is the C-terminal amino acid, x=8-11 such that Rx can befrom the eighth to the eleventh amino acid residue from Rn, R2 or R3 andRx are primary anchor residues of a motif or a supermotif, and (b)producing a polypeptide comprising an analog, said analog comprising aformula (B) identical to said formula (A) except one or morenonconservative amino acid substitutions at R3 and/or R5 and/or R7.

Thus, in some aspects, R7 is Tyr in formula (A), and said analogcomprises a formula (B) identical to said formula (A) except that R7 isGly, Glu, or Asp.

The second Class I epitope described above is generically referred to asa “heteroclitic analog” or an “analog.”

In a preferred embodiment, the heteroclitic analog exhibits at leastabout 50% increased potency for a specific T-cell compared to thecorresponding wildtype Class I epitope. The analog may contain only onesubstitution, or may contain two or three, and the substitution may beconservative or semi-conservative or, in the case of a B7 superfamilyepitope, non-conservative. The heteroclitic analog may induce both Th1and Th2 cytokines when bound by an HLA Class I molecule and contactedwith the relevant cytotoxic T-cell. Preferably, the Class I epitopecomprises an HLA supermotif selected from the group consisting of A1,A2, A3, A24, B7, B27, B44, B58 and B62, more preferably, the Class Iepitope comprises an A2 supermotif or a B7 supermotif, most preferably,an A2.1 motif (e.g. an A*0201), or a B7 motif (e.g. a B*0702 motif).

The class I epitope may be from a viral antigen, a tumor-associatedantigen, a parasitic antigen, a bacterial antigen or a fungal antigen.

The supermotif may be A1, wherein R2 is a primary anchor residue and iseither T, I, L, V, M or S, and Rx is either F, W, or Y.

The supermotif may be A2, wherein R2 is a primary anchor residue and iseither L, I, V, M, A, T, or Q, and Rx is I, V, M, A, T, or L.

The supermotif may be A2.1, wherein R2 is a primary anchor and is eitherL, M, V, Q, I, A, or T, and Rx is either V, L, I, M, A, or T.

The supermotif may be A3, wherein R2 is a primary anchor residue and iseither V, S, M, A, T, L, or I, and Rx is R or K.

The supermotif may be A24, wherein R2 is a primary anchor residue and iseither Y, f, W, I, V, L, M, or T, and Rx is either F, I, Y, W, L, or M.

The supermotif may be B7, wherein R2 is a primary anchor residue and isP and Rx is either V, I, L, F, M, W, Y, or A.

The invention also provides methods of inducing a human cytotoxic T cellresponse against a preselected Class I peptide epitope, the methodcomprising providing the heteroclitic analog described above; andcontacting a human CTL with the heteroclitic analog.

In some aspects, the step of contacting is carried out in vitro. In someaspects, the step of contacting is carried out by administering to asubject a nucleic acid molecule comprising a sequence encoding theheteroclitic analog peptide epitope.

The invention also provides polypeptides produced by the methoddescribed above. The invention is also directed to peptides, e.g.,polypeptides, comprising the heteroclitic analog epitopes which areobtainable by the method described above. In particular, and preferably,such peptides include those where the epitope (e.g., analog) consists ofan amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:49, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53. The peptide maycontain 9-20 amino acids, preferably 9-16, more preferably 9-15, but mayalso contain only a total of 9, 10, 11, 12, 13 or 14 amino acids. Thedefined heteroclitic analog epitopes may be included in a longerpolypeptide or protein which is a homopolymer of the same epitope (e.g.,analog) or a heteropolymer which contains a variety of such epitopes(e.g., analogs) or the heteroclitic analog epitope in combination withwildtype epitopes. These peptides and proteins may be included incompositions which are designed for pharmaceutical use.

The peptides or heteropolymers or homopolymers containing theheteroclitic analog epitopes may be combined with other components toenhance further or modulate their activity in eliciting an immuneresponse. These additional varieties may be covalently bound ornon-covalently included in a mixture.

Thus, the polypeptide may comprise a T helper peptide, a spacer orlinker amino acid, a carrier, may be linked to a lipid, may comprise afusion protein, may comprise a homopolymer, a heteropolymer, and/or maycomprise one or more second epitopes or second analogs.

Further, the heteroclitic analog epitope may be admixed or joined to aCTL epitope, or to an HTL epitope, especially where HTL epitope is apan-DR binding molecule. A composition containing the heterocliticanalog epitope may further comprise a liposome, wherein the epitope ison or within the liposome, or the epitope may be joined to a lipid. Theheteroclitic epitope may be bound to an HLA heavy chain,β2-microglobulin, and strepavidin complex, whereby a tetramer is formed.In addition, the heteroclitic epitope (e.g., a polypeptide comprising ananalog) may be modified in a composition which comprises an antigenpresenting cell, wherein the epitope (e.g., a polypeptide comprising ananalog) is on or within the antigen presenting cell, wherein the epitope(e.g., a polypeptide comprising an analog) is bound to an HLA moleculeon the antigen presenting cell. Thus, when a cytotoxic lymphocyte (CTL)that is restricted to the HLA molecule is present, a receptor of the CTLbinds to a complex of the HLA molecule and the epitope (e.g., apolypeptide comprising an analog). The antigen presenting cell may be adendritic cell. The composition may also simply comprise an HLAmolecule, wherein the peptide containing the epitope (e.g., apolypeptide comprising an analog) is bound by the HLA molecule. Thecomposition may also comprise a label—e.g., biotin, a fluorescentmoiety, a non-mammalian sugar, a radiolabel or a small molecule to whicha monoclonal antibody binds.

The compositions described are useful in eliciting an immune responseagainst the corresponding wildtype epitope. Typically, the heterocliticanalog is included in such compositions which will further containsuitable excipients. The active component heteroclitic epitopes (e.g., apolypeptide comprising an analog)s may be present in unit dosage form.Compositions useful in treating subjects may also comprise nucleic acidmolecules that encode the peptides described above optionally includingcontrol sequences for their expression.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIGS. 1A-1D. FIGS. 1A and 1B represent the results of testing a panel ofanalogs of CEA.691 and MAGE3.112 respectively for ability to induce IFNγproduction in the corresponding CTL. FIGS. 1C and 1D are thecorresponding dose response curves for CEA.691 and MAGE3.112heteroclitic analogs respectively.

FIGS. 2A-2D. FIGS. 2A, 2B and 2C show the results of testing panels ofanalogs of MAGE2.157, HIVPol.476, and HBVPol.455 epitope analogs withrespect to the ability of these analogs to induce IFNγ production in thecorresponding CTLs. FIG. 2D is the relevant dose response curve for thesuccessful HIVPol.476 analogs.

FIGS. 3A and 3B show dose response curves of heteroclitic analogs ofMAGE2.157 in comparison to wildtype with regard to their ability toinduce IFNγ production or IL10 production from the appropriate CTLs.

FIGS. 4A and 4B are the dose response curves for wildtype and aheteroclitic analog of HIVPol.476 to produce IFNγ and IL10 inappropriate CTLs.

FIG. 5 shows the results of testing a panel of potential heterocliticanalogs of the epitope p53.149M2 with respect to IFNγ production fromappropriate CTLs.

FIGS. 6A and 6B are the corresponding dose response curves forproduction of IFNγ and IL10 by successful heteroclitic analogs ofp53.149M2.

FIG. 7 shows the results of testing a panel of potential analogs ofp53.Mu184 epitope for IFNγ production in CTLs.

FIG. 8 shows the dose response curve for wildtype and two successfulheteroclitic analogs of p53.Mu184 with respect to IFNγ production.

FIGS. 9A-9D show the cross-reactivity of heteroclitic analogs withregard to the corresponding wildtype epitope. In FIGS. 9A and 9B, IFNγproduction is plotted as a function of concentration using stimulationby the immunizing peptide. FIGS. 9C and 9D show the correspondingresults when wildtype epitope is used as the stimulant as opposed to theheteroclitic analog used for the initial induction of CTL.

FIG. 10 shows the IFNγ release with respect to stimulation by p53.261and its heteroclitic analogs.

FIG. 11 shows Elispot results with respect to various heterocliticanalogs.

FIGS. 12A-12C show the results of stimulation of CTL activity againstendogenous peptide using various heteroclitic analogs.

FIGS. 13A-13B show the results of testing a panel of potentialheteroclitic analogs of the epitope MAGE2.170 with respect to IFNγproduction from appropriate CTLs. Single residue substitutions, eitherconservative/semi-conservative or non-conservative in nature, wereintroduced in the MAGE2.170 epitope at every non-MHC anchor position.Peptide analogs were screened for their capacity to stimulate a humanCTL line specific for the MAGE2.170 wildtype epitope at two peptidedoses. CTL responses were measured by stimulating CTL in vitro withpeptide at the two indicated doses in the presence of GM3107 tumor cellsas APC. IFNγ production of stimulated CTL was measured by ELISA. Thex-axis shows the substituted residue (underlined residues denotenon-conservative substitutions) and each response bar corresponds to thestimulatory activity of that analog. The native residue at the givenposition in the MAGE2.170 epitope is shown at the top of each panel.

FIG. 14 shows dose response curves of heteroclitic analogs of MAGE2.170in comparison to wildtype with regard to their ability to induce IFNγ.Analogs with hyperstimulatory activity identified in the initialscreening assay were tested for CTL stimulation in a peptide dosetitration. Each analog was tested in a dose titration against a humanCTL line specific for the wildtype epitope and GM3107 cells as APC. IFNγrelease was measured with an ELISA.

MODES OF CARRYING OUT THE INVENTION 1. Overview

The present invention relates to methods of designing heterocliticanalogs that bind to HLA Class I molecules. “Heteroclitic analogs,” asdescribed herein, are peptides comprising epitopes with increasedpotency for a specific T cell, as measured by increased responses to agiven dose, or by a requirement of lesser amounts to achieve the sameresponse as a homologous Class I peptide. The methods of the inventionare useful to modify any Class I peptide, particularly those associatedwith human cancers and precancerous conditions, and from infectiousagents such as viruses, bacteria, fungi, and protozoal parasites.

Importantly, the phenomenon of heteroclicity applies across HLAmolecules that bind a particular Class I peptide. For example, aheteroclitic analog peptide bearing the A2 supermotif is heteroclitic(i.e., has higher potency) across all HLA molecules in the HLA-supertype(e.g., A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, etc.; seeTable 5). Similarly, a heteroclitic analog peptide bearing the B7supermotif is heteroclitic across all HLA molecules in the HLA-supertype(e.g., B*0702, B*0703, B*0704, B*0705, B*1508, B*3501, B*3502, B*3503,B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101, B*5102, B*5103,B*5104, B*5105, B*5301, B*5401, B*5501, B*5502, B*5601, B*5602, B*6701,B*7801, etc.; see Table 5). Thus, a heteroclitic analog peptide bearinga different sequence motif (e.g., A1, A2, A3, A24, B7, B27, B44, B58,B62, etc.) induces a more potent immune response across all HLAmolecules within their specific HLAsuperfamily.

Applicants have found specific rules for designing heteroclitic analogswhich enhance the immune response to the corresponding wildtype epitope.These rules are applicable with respect to epitopes bearing motifs orsupermotifs which bind to HLA molecules encoded by any Class I allele.By using these rules, it is possible to enhance the immunogenicity,therefore, of any “wildtype” or “native” Class I epitope.

Briefly, the rules state that the wildtype Class I epitope is modifiedby substituting a conservative or semi-conservative amino acid asposition 3 and/or 5 and/or 7 of the epitope. For B7 superfamilyepitopes, the rule states that the wildtype Class I epitope (i.e., theB7 superfamily epitope) is modified by substituting a conservative orsemi-conservative or non-conservative amino acid as position 3 and/or 5and/or 7 of the epitope. The nature of the conservative orsemi-conservative or non-conservative amino acid to be substituted isdefined by the description in Preparation B hereinbelow, the results ofwhich are summarized in Table 2. Thus, by consulting Table 2, one candetermine suitable candidates for substitution at these positions. Asshown in Table 2, each of the amino acids shown across the top of thetable bears a numerically defined relationship to the remaining 19genetically encoded amino acids. The lower the index, the higher theconservation; the same amino acid will have a similarity assignment of1.0; maximally different amino acids will have similarity assignmentsapproaching 20. Using the method set forth in Preparation B, amino acidswhich are not gene-encoded can also be assigned similarity indices andcan be classified with respect to any natively occurring amino acid asconservative or semi-conservative (or non-conservative).

Heteroclitic analog peptides of the invention are particularly useful toinduce an immune response against antigens to which a subject's immunesystem has become tolerant. Human subjects are particularly preferred,but the methods can also be applied to other mammals such as laboratorymice, taking account of the corresponding HLA motifs with regard tothese subjects. Tolerance refers to a specific immunologicnonresponsiveness induced by prior exposure to an antigen. Tolerance canbe overcome by identifying a particular Class I peptide epitope to whicha patient is tolerant, modifying the peptide epitope sequence accordingto the methods of the invention, and inducing an immune response thatcross-reacts against the tolerized epitope (antigen). Overcomingtolerance is particularly desirable, for example, when the immune systemof the subject is tolerant of a viral or tumor-associated antigen, thelatter antigens being often over-expressed self-proteins as aconsequence of cell transformation.

To determine rules for designing heteroclitics, several different CTLlines were screened for reactivity against panels of analogs.Modification of T cell stimulatory capacity was achieved with noalternation of the primary MHC anchors.

The wildtype epitopes include tumor epitopes derived from self antigensthat are specifically up-regulated in epithelial cell cancers and havebeen shown to be immunogenic. Viral epitopes used, such as those fromthe polymerase genes of the HIV and HBV, have been shown to beimmunogenic as well.

The rules described herein provide a basis to design heterocliticanalogs, drastically reducing the screening otherwise required and areextremely useful in designing epitope-based vaccines for cancer andinfectious diseases.

In the examples set forth below, 17% of the total analogs screened(which fit the heteroclicity rules disclosed herein) were heteroclitic(16/95). This is significant for two reasons: first, the efficiency ofdetecting heteroclitics increased from 2.2% to 17% by employing analogsthat follow the rules of heteroclitic substitution; second, the numberof peptides which need to be synthesized is reduced dramatically fromabout a 100 analogs per epitope to about I5 analogs per epitope, makingthe process cost effective and amenable to high throughput. Through theapplication of the heteroclitic substitution rules of the invention, theefficiency of generating heteroclitic analogs was increased nearly 100to 1000-fold, from 0.2% (4 identified from screening of 233 CEA.691 andMAGE3.112 analogs) to 33% (3 identified by screening of 9 predictedanalogs). The latter frequency may be a gross underestimate since only 4of 6 analogs showing potential heteroclitic activity in initial assayswere subjected to further analysis.

Previous studies showed that modulation of T cell responses byheteroclitic analogs involved TCR contact residues (Byrne, et al., J.Immunol. 51:682 (1984), McMichael, et al., K. England. J. Med. 309:13(1983), Zugel, et al., J. Immunol. 161:1705 (1998), Rivoltini, et al.,Cancer Research 59:301 (1999)), but the present study did not find this.For example, for the CEA.691 epitope, the TCR contact residue isposition 8, while heteroclicity was observed with analog substitutionsat positions 3 and 5. While not intending to be bound by any theory,alteration of MHC binding may be a mechanism. Binding analyses performedon the analogs indicated that there is an alteration in MHC binding forthe better or worse in a majority of cases (80%). Out of the 13 analogswhich were tested for HLA-A2 binding, ten analogs had alteration in MHCbinding, with six analogs binding better than wildtype peptides and fouranalogs that bound worse than wildtype, but still generated asubstantially increased biological response. Some studies modify primaryMHC anchor residues in order to increase MHC binding (this approach hasbeen used by some groups to generate analogs (Pfeiffer, et al., J. Exp.Med. 181:1569 (1995), Valmori, et al., J. Immunol. 160:1750-1758 (1998),Parkhurst, et al., J. Immunol. 157:2539 (1996)). Increased biologicalresponses without changing primary TCR contact residues or primary MHCanchor residues was observed in this study. Since increased responseswere mediated with alteration in MHC binding, it is postulated that theeffect may be mediated by changing secondary anchor positions. Moreevidence supporting this comes from the finding that heterocliticsubstitutions occur at odd numbered positions (3, 5, 7) in the middle ofthe peptide. All these positions 3, 5, and 7 have been shown to besecondary anchor positions for binding to the HLA-A2 molecule (Ruppert,et al., Cell 74:929 (1993), Madden, Annu. Rev. Immunol. 13:587-622(1995)).

Two of these positions (3 and 7) have been shown to be secondary anchorpositions for binding to HLA-A2.1 molecule by several groups (Ruppert,et al., Cell 74:929 (1993), Madden, Annu. Rev. Immunol. 13:587-622(1995)). Alteration of such secondary anchor positions can translateinto T cell recognition differences (Valmori, et al., J. Immunol.160:1750 (1998); Davis, et al., Annu. Rev. Immunol. 16:523 (1998)),however in these studies T cell recognition differences were associatedwith changes in MHC binding and no rules were defined for the kinds ofamino acid substitutions involved in obtaining heteroclicity. Themechanism by which such a translation from changing secondary anchors tochange in T cell recognition takes place is currently unclear. However,some models suggest that changes in the way residues at secondary anchorpositions engage the MHC may lead to alteration in the orientation orincreased flexibility of TCR contact residues, resulting in enhancementof the binding of these analogs to the TCR (Kersh, et al., J. Exp. Med.184:1259 (1996), Evavold, et al., J. Immunol. 148:347 (1992), Alam, etal., Immunity 10:227 (1999), Hampl, et al., Immunity 7:379-85 (1997)).Also, some previous studies implied that modulation of T cell responsesby heteroclitic analogs directly involve main TCR contact residues(Zaremba, et al., Cancer Research 57:4570 (1997), Loftus, et al., CancerResearch 58:2433 (1998), Dressel, et al., J. Immunol. 159:4943 (1997)).This finding, however, is not corroborated by the current systematicanalysis. The enhanced T cell recognition against analogs identified inthe present study is not likely due to increases in MHC bindingcapacity, though increased binding is likely to play an important rolein the case of analogs in which primary anchor positions have beenoptimized. The present study suggests that heteroclitic analogs are mostlikely generated by subtle alterations in conformation rather than bygross alterations of TCR or MHC binding capacity.

Differential regulation of production of Th1 or Th2 cytokines was notobserved. Instead, the present data suggested that the heterocliticanalogs increased the production of both Th1 and Th2 responses, althoughthe magnitude and kinetics of the increase may be different. In fact,some groups (Nicholson, et al., Int. Immunol. 12 (2):205-13 (2000),Parkhurst, et al., J. Immunol. 157:2539 (1996)) have recently reportedsuch overall stimulation by peptide analogs. This is attributable to astronger TCR signal induced by analogs, though the mechanism of suchoverall stimulation remains to be elucidated.

The efficacy of heteroclitic analogs in vivo using relevant tumor modelsor models in which tolerance to self antigens exists is evaluated.Accordingly, it is found that immunization with heteroclitic analogs isa more effective and efficient strategy for vaccination against tumorswhere raising effective CTLs has so far proved to be a challenge.

To summarize, in a set of experiments, applicants have identifiedheteroclitic analogs of a number of different HLA-A2.1-restricted CTLepitopes of cancer and viral origin. The relevant wildtype epitopes areshown in Table 1. All these epitopes have been shown to be immunogenicin our earlier reports (Kawashima, et al., Human Immunology 59:1-14(1998), Ishioka, et al., J. Immunol. 162 (7):3915-25 (1999)). In initialexperiments, the antigenicity of 233 analogs of the CEA.691 andMAGE3.112 CTL epitopes was investigated. The nature of the fourheteroclitic analogs identified suggested that heterocliticsubstitutions involved conservative substitutions at positions 3, 5 and7. This hypothesis, was tested in a subsequent study involving threeadditional epitopes MAGE2.157, HIVPol.476, and HBVPol.455. All of theheteroclitic analogs thus identified conformed to the rules proposed,namely that heteroclitic analogs were associated with conservative orsemi-conservative substitutions at positions 3, 5 and/or 7.

To more closely mimic the clinical application of heteroclitic analogsin cancer immunotherapy, the murine epitope, p53.261 was also modified.A partial state of T cell tolerance has been reported for this epitope(Theobald, et al., Proc. Natl. Acad. Sci. 92:11993-11997 (1995),Theobald, et al., J. Exp. Med., 185 (5):833-841 (1997)). Four out ofnine predicted p53.261 analogs were found to induce strongeranalog-specific CTL responses in vivo compared to the CTL responsesinduced by the native peptide. More significantly, when thecross-reactivity of the CTL raised by immunization with heterocliticanalogs was analyzed, three p53.261 analogs induced CTL which respondedvigorously against the native p53.261 epitope. Finally, the relevance ofthese findings for human CTL was addressed by demonstrating thatheteroclitic analogs of the MAGE3.112 epitope are immunogenic for humanT cells in vitro. The resulting CTL can recognize wildtype naturallyprocessed antigen in the form of tumor cell lines.

The studies presented herein demonstrate that heteroclicity is a globalphenomenon, as heteroclitic analogs were identified for all the epitopesstudied. In addition, the present application shows that it is possibleto detect heteroclitic analogs both in clonal T cell populations (as hasbeen described earlier studies) as well as in bulk T cell populationsfollowing in vivo immunization. Moreover, it is demonstrated herein thatheteroclicity (both in the HLA A2.1 system as well as for other Class Isupermotifs) is associated with discrete structural features which allowrational prediction of heteroclicity.

It is demonstrated, further that p53.261 heteroclitic analogs induceCTLs with higher avidity and also induced these cells in greater numbers(precursor frequency) than those induced with wildtype peptide;heteroclitic CTL induction in vivo, and its application to breaking Tcell tolerance is demonstrated.

The heteroclitic analogs were effective in raising bulk populations ofspecific T cells following in vivo immunization. Polyclonal responsesthat bear TCR from multiple TCR genes, are more efficacious in resolvingdisease states in a clinical setting. Finally, the ability to generatehigh precursor frequencies of CTL possessing strong cross-reactiveavidity against wildtype epitope is important in instances whereeffective CTL responses against epitopes, normally tolerant to theimmune system, are required.

In another set of experiments, applicants identified heterocliticanalogs of the B7 superfamily epitope MAGE2.170 (shown in Table 1). LikeA2 heteroclitic epitopes, heteroclitic analogs of the B7 superfamilyepitope could be generated by introducing substitutions at an odd-numberposition in the middle of the peptide (position 7). The nature of thesubstitutions for the MAGE2.170 epitope were eitherconservative/semi-conservative (the Y→H and Y→M substitutions) ornon-conservative (the Y→E, Y→G, and Y→D substitutions) compared to thenative residue (Table 5). Thus, the observation that non-conservativesubstitutions can result in heteroclitic analogs for the MAGE2.170 CTLepitope indicate a partially overlapping substitution pattern than thatobserved with A2 superfamily epitopes.

2. Definitions

With regard to a particular amino acid sequence, an “epitope” is a setof amino acid residues which is involved in recognition by a particularimmunoglobulin, or in the context of T cells, those residues necessaryfor recognition by T cell receptor proteins when presented in thecontext of an HLA encoded by the Major Histocompatibility Complex (MHC).In an immune system setting, in vitro or in vivo, an epitope is thecollective features of a molecule, such as primary, secondary andtertiary peptide structure, and charge, that together form a siterecognized by an immunoglobulin, T cell receptor or HLA molecule.Throughout this disclosure epitope and peptide are often usedinterchangeably. It is to be appreciated, however, that isolated orpurified protein or peptide molecules larger than and comprising anepitope of the invention are still within the invention.

A “Class I epitope” refers to a peptide that binds to a Class I HLAmolecule. As described herein, a Class I epitope is typically about 8 toabout 13 amino acids in length. Binding to the HLA molecule is primarilycontrolled by two primary anchor residues, one of which is at theC-terminus of the epitope and the other of which is at positions 2 or 3.Binding may be aided also by one or more secondary anchor residues. Forthe convenience of the reader, various primary HLA Class I bindinganchors are set forth in Table 3. The pattern of anchors is referred toas a “motif.” A “supermotif” is a peptide binding specificity shared byHLA molecules encoded by two or more HLA alleles. Preferably, asupermotif-bearing peptide is recognized with high or intermediateaffinity (as defined herein) by two or more HLA antigens. Examples ofClass I supermotifs include, e.g., A1, A2, A3, A24, B7, B27, B44, B58and B62.

Throughout this disclosure, “binding data” results are often expressedin terms of “IC₅₀'s.” IC₅₀ is the concentration of peptide in a bindingassay at which 50% inhibition of binding of a reference peptide isobserved. Given the conditions in which the assays are run (i.e.,limiting HLA proteins and labeled peptide concentrations), these valuesapproximate Kd values. Assays for determining binding are described indetail, e.g., in PCT publications WO 94/20127 and WO 94/03205,incorporated herein by reference. It should be noted that IC₅₀ valuescan change, often dramatically, if the assay conditions are varied, anddepending on the particular reagents used (e.g., HLA preparation, etc.).For example, excessive concentrations of HLA molecules will increase theapparent measured IC₅₀ of a given ligand. Alternatively, binding isexpressed relative to a reference peptide. Although as a particularassay becomes more, or less, sensitive, the IC₅₀'s of the peptidestested may change somewhat, the binding relative to the referencepeptide will not significantly change. For example, in an assay rununder conditions such that the IC₅₀ of the reference peptide increases10-fold, the IC₅₀ values of the test peptides will also shiftapproximately 10-fold. Therefore, to avoid ambiguities, the assessmentof whether a peptide is a good, intermediate, weak, or negative binderis generally based on its IC₅₀, relative to the IC₅₀ of a standardpeptide. Binding may also be determined using other assay systems knownin the art.

The designation of a residue position in an epitope as the “carboxyl orC-terminus” refers to the residue position at the end of the epitopewhich is nearest to the carboxyl terminus of a peptide, which isdesignated using conventional nomenclature as defined below. The“C-terminus” of the epitope may or may not actually correspond to theend of the peptide or polypeptide.

The designation of a residue position in an epitope as “N-terminus” or“amino-terminal position” refers to the residue position at the end ofthe epitope which is nearest to the N-terminus of a peptide, which isdesignated using conventional nomenclature as defined below. The“N-terminus” of the epitope may or may not actually correspond to theend of the peptide or polypeptide.

A “computer” or “computer system” generally includes: a processor; atleast one information storage/retrieval apparatus such as, for example,a hard drive, a disk drive or a tape drive; at least one input apparatussuch as, for example, a keyboard, a mouse, a touch screen, or amicrophone; and display structure. Additionally, the computer mayinclude a communication channel in communication with a network. Such acomputer may include more or less than what is listed above.

As used herein amino acids that are “conserved” or “conservative,” and“semi-conserved” or “semi-conservative,” and “non-conserved” or“non-conservative” are defined in accordance with Preparation B and setforth in Table 2.

As used herein, “high affinity” with respect to HLA Class I molecules isdefined as binding with an IC₅₀, or K_(D) value, of 50 nM or less;“intermediate affinity” is binding with an IC₅₀ or K_(D) value ofbetween about 50 and about 500 nM. “High affinity” with respect tobinding to HLA Class II molecules is defined as binding with an IC₅₀ orK_(D) value of 100 nM or less; “intermediate affinity” is binding withan IC₅₀ or K_(D) value of between about 100 and about 1000 nM.

An “immunogenic peptide” or “peptide epitope” is a peptide thatcomprises an allele-specific motif or supermotif such that the peptidewill bind an HLA molecule and induce a CTL and/or HTL response. Thus,immunogenic peptides of the invention are capable of binding to anappropriate HLA molecule and thereafter inducing a cytotoxic T cellresponse, or a helper T cell response, to the antigen from which theimmunogenic peptide is derived.

The phrases “isolated” or “biologically pure” refer to material that issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment.

A “PanDR binding peptide” is a member of a family of molecules thatbinds more that one HLA Class II DR molecule (e.g., PADRE™ peptide,Epimmune Inc., San Diego, Calif.). The pattern that defines the PADRE™family of molecules can be thought of as an HLA Class II supermotif.Peptides comprising the pattern found in PADRE™ molecules bind to mostHLA-DR molecules and stimulate in vitro and in vivo human helper Tlymphocyte (HTL) responses.

“Pharmaceutically acceptable” refers to a generally non-toxic, inert,and/or physiologically compatible composition.

3. Peptides of the Invention

Peptides in accordance with the invention can be prepared synthetically,by recombinant DNA technology or chemical synthesis, or from naturalsources such as native tumors or pathogenic organisms. Peptide epitopesmay be synthesized individually or as polyepitopic peptides. Althoughthe peptide will preferably be substantially free of other naturallyoccurring host cell proteins and fragments thereof, in some embodimentsthe peptides may be synthetically conjugated to native fragments orparticles.

HLA Class I peptides are well known in the art and are defined aspeptides that bind to MHC Class I molecules. The peptides in accordancewith the invention can be a variety of lengths, and either in theirneutral (uncharged) forms or in forms which are salts. The peptides inaccordance with the invention are either free of modifications such asglycosylation, side chain oxidation, or phosphorylation; or they containthese modifications, subject to the condition that modifications do notdestroy the biological activity of the peptides as described herein.

Class I epitopes that serve as the corresponding “wildtype” can bederived from any proteinaceous source. For example, the Class I peptidescan be derived from viral antigens, tumor-associated antigens, parasiticantigens, bacterial antigens or fungal antigens. In some preferredaspects of the invention, the Class I peptide(s) are derived fromantigens for which a the immune system of a subject has developed atolerance, i.e., a specific immunologic nonresponsiveness induced byprior exposure to an antigen.

Thus, heteroclitic analogs based on a number of potential targetepitopes can be used in the present invention. Examples of suitabletumor-associated antigens include prostate specific antigens (PSA),melanoma antigens MAGE 1, MAGE 2, MAGE 3, MAGE-11, MAGE-A10, as well asBAGE, GAGE, RAGE, MAGE-C1, LAGE-1, CAG-3, DAM, MUC1, MUC2, MUC18,NY-ESO-1, MUM-1, CDK4, BRCA2, NY-LU-1, NY-LU-7, NY-LU-12, CASP8, RAS,KIAA-2-5, SCCs, p53, p73, CEA, Her 2/neu, Melan-A, gp100, tyrosinase,TRP2, gp75/TRP1, kallikrein, prostate-specific membrane antigen (PSM),prostatic acid phosphatase (PAP), prostate-specific antigen (PSA),PT1-1, β-catenin, PRAME, Telomerase, FAK, cyclin D1 protein, NOEY2,EGF-R, SART-1, CAPB, HPVE7, p15, Folate receptor CDC27, PAGE-1, andPAGE-4. Examples of suitable infectious disease-associated antigensinclude hepatitis B core and surface antigens (HBVc, HBVs), hepatitis Cantigens, Epstein-Barr virus antigens, human immunodeficiency virus(HIV) antigens and human papilloma virus (HPV) antigens, Mycobacteriumtuberculosis and Chlamydia. Examples of suitable fungal antigens includethose derived from Candida albicans, Cryptococcus neoformans,Coccidoides spp., Histoplasma spp, and Aspergillus fumigatis. Examplesof suitable protozoal parasitic antigens include those derived fromPlasmodium spp., including P. falciparum, Trypanosoma spp., Schistosomaspp., Leishmania spp and the like.

The epitopes that may be used as wildtype sequences to which the rulesof the invention are applied to construct corresponding heterocliticanalogs can be found corresponding to any Class I epitope. For anydesired antigen, such as those set forth above, the motif associatedwith a particular Class I allele can be used as a guide to determine thepositions in the amino acid sequence of the antigen wherein such anepitope would reside. This determination can be done visually or,preferably, using computer technology and associated software. Thus, forexample, by recognition of the A3 supermotif as containing, for example,valine in position 2 and arginine at the C-terminus, the amino acidsequence of any desired antigen can be surveyed for epitopes bearingthis motif. That epitope can then be modified according to the rules setforth in the present invention to obtain the desired analogs.

When possible, it may be desirable to optimize HLA Class I bindingepitopes of the invention, such as can be used in a polyepitopicconstruct, to a length of about 8 to about 13 amino acid residues, often8 to 11, preferably 9 to 10. Preferably, the peptide epitopes arecommensurate in size with endogenously processed pathogen-derivedpeptides or tumor cell peptides that are bound to the relevant HLAmolecules, however, the identification and preparation of peptides thatcomprise epitopes of the invention can also be carried out using thetechniques described herein.

In alternative embodiments, epitopes of the invention can be linked as apolyepitopic peptide, or as a minigene that encodes a polyepitopicpeptide.

In another embodiment, it is preferred to identify native peptideregions that contain a high concentration of Class I epitopes and/orClass II epitopes. Such a sequence is generally selected on the basisthat it contains the greatest number of epitopes per amino acid length.It is to be appreciated that epitopes can be present in a nested oroverlapping manner, e.g., a 10 amino acid long peptide could contain two9 amino acid long epitopes and one 10 amino acid long epitope; uponintracellular processing, each epitope can be exposed and bound by anHLA molecule upon administration of such a peptide. This larger,preferably multi-epitopic, peptide can be generated synthetically,recombinantly, or via cleavage from the native source.

The peptides of the invention can be prepared in a wide variety of ways.For the preferred relatively short size, the peptides can be synthesizedin solution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. (See, for example,Stewart & Young, SOLID PHASE PEPTIDE SYNTHESIS, 2D. ED., Pierce ChemicalCo., 1984). Further, individual peptide epitopes can be joined usingchemical ligation to produce larger peptides that are still within thebounds of the invention.

Alternatively, recombinant DNA technology can be employed wherein anucleotide sequence which encodes an immunogenic peptide of interest isinserted into an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression. These procedures are generally known in the art, asdescribed generally in Sambrook, et al., MOLECULAR CLONING, A LABORATORYMANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Thus,recombinant polypeptides which comprise one or more peptide sequences ofthe invention can be used to present the appropriate T cell epitope.

The nucleotide coding sequence for peptide epitopes of the preferredlengths contemplated herein can be synthesized by chemical techniques,for example, the phosphotriester method of Matteucci, et al., J. Am.Chem. Soc. 103:3185 (1981). Peptide analogs can be made simply bysubstituting the appropriate and desired nucleic acid base(s) for thosethat encode the native peptide sequence; exemplary nucleic acidsubstitutions are those that encode an amino acid defined by themotifs/supermotifs herein. The coding sequence can then be provided withappropriate linkers and ligated into expression vectors commonlyavailable in the art, and the vectors used to transform suitable hoststo produce the desired fusion protein. A number of such vectors andsuitable host systems are now available. For expression of the fusionproteins, the coding sequence will be provided with operably linkedstart and stop codons, promoter and terminator regions and usually areplication system to provide an expression vector for expression in thedesired cellular host. For example, promoter sequences compatible withbacterial hosts are provided in plasmids containing convenientrestriction sites for insertion of the desired coding sequence. Theresulting expression vectors are transformed into suitable bacterialhosts. Of course, yeast, insect or mammalian cell hosts may also beused, employing suitable vectors and control sequences.

Analogs of the present invention may include peptides containingsubstitutions to modify the physical property (e.g., stability orsolubility) of the resulting peptide. For example, peptides may bemodified by the substitution of a cysteine (C) with α-amino butyricacid. Due to its chemical nature, cysteine has the propensity to formdisulfide bridges and sufficiently alter the peptide structurally so asto reduce binding capacity. Substituting α-amino butyric acid for C notonly alleviates this problem, but actually improves binding andcrossbinding capability in certain instances. Substitution of cysteinewith α-amino butyric acid may occur at any residue of a peptide epitope,i.e. at either anchor or non-anchor positions.

Modified peptides that have various amino acid mimetics or unnaturalamino acids are particularly useful, as they tend to manifest increasedstability in vivo. Such analogs may also possess improved shelf-life ormanufacturing properties. More specifically, non-critical amino acidsneed not be limited to those naturally occurring in proteins, such asL-α-amino acids, or their D-isomers, but may include non-natural aminoacids as well, such as amino acids mimetics, e.g. D- or L-naphylalanine;D- or L-phenylglycine; D- or L-2-thieneylalanine; D- or L-1,-2,3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-ρ-fluorophenylalanine; D- or L-ρ-biphenylphenylalanine; D- orL-ρ-methoxybiphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylalanines, where the alkyl group can be a substituted orunsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl,iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromaticrings of a nonnatural amino acid include, e.g., thiazolyl, thiophenyl,pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridylaromatic rings.

Peptide stability can be assayed in a number of ways. For instance,peptidases and various biological media, such as human plasma and serum,have been used to test stability. See, e.g., Verhoef, et al., Eur. J.Drug Metab. Pharmacokinetics 11:291 (1986). Half life of the peptides ofthe present invention is conveniently determined using a 25% human serum(v/v) assay. The protocol is generally as follows: Pooled human serum(Type AB, non-heat inactivated) is delipidated by centrifugation beforeuse. The serum is then diluted to 25% with RPMI-1640 or another suitabletissue culture medium. At predetermined time intervals, a small amountof reaction solution is removed and added to either 6% aqueoustrichloroacetic acid (TCA) or ethanol. The cloudy reaction sample iscooled (4° C.) for 15 minutes and then spun to pellet the precipitatedserum proteins. The presence of the peptides is then determined byreversed-phase HPLC using stability-specific chromatography conditions.

4. Class I Motifs

In the past few years, evidence has accumulated to demonstrate that alarge fraction of HLA Class I molecules can be classified into arelatively few supertypes, each characterized by largely overlappingpeptide binding repertoires, and consensus structures of the mainpeptide binding pockets. Thus, peptides of the present invention areidentified by any one of several HLA-specific amino acid motifs (see,e.g., Tables 3-4), or if the presence of the motif corresponds to theability to bind several allele-specific HLA antigens, a supermotif. TheHLA molecules that bind to peptides that possess a particular amino acidsupermotif are collectively referred to as an HLA “supertype.”

For the convenience of the reader, the peptide motifs and supermotifsdescribed below, and summarized in Tables 3-4, provide guidance for theidentification and use of peptide epitopes in accordance with theinvention. This will permit identification of candidate wildtypeepitopes corresponding to various Class I motifs different from thoseillustrated in the examples below or epitopes bearing those illustratedbelow but in different antigens in order to apply the rules set forthherein to construct analogs.

Heteroclitic analogs can be designed according to the methods of theinvention from a peptide, without regard to the motif or supermotif towhich the peptide belongs. The primary anchor residues of the HLA ClassI peptide epitope supermotifs and motifs delineated below are summarizedin Table 3. The HLA Class I motifs set out in Table 4 are those mostparticularly relevant to the invention claimed here. Allele-specific HLAmolecules that comprise HLA Class I supertype families are listed inTable 5. In some cases, peptide epitopes may be listed in both a motifand a supermotif. The relationship of a particular motif and respectivesupermotif is indicated in the description of the individual motifs.

i. HLA-A1 Supermotif

The HLA-A1 supermotif is characterized by the presence in peptideligands of a small (T or S) or hydrophobic (L, I, V, or M) primaryanchor residue in position 2, and an aromatic (Y, F, or W) primaryanchor residue at the C-terminal position of the epitope. Thecorresponding family of HLA molecules that bind to the A1 supermotif(i.e., the HLA-A1 supertype) is comprised of at least A*0101, A*2601,A*2602, A*2501, and A*3201 (see, e.g., DiBrino, M. et al., J. Immunol.151:5930, 1993; DiBrino, M. et al., J. Immunol. 152:620, 1994; Kondo, A.et al., Immunogenetics 45:249, 1997). Other allele-specific HLAmolecules predicted to be members of the A1 superfamily are shown inTable 5.

ii. HLA-A2 Supermotif

Primary anchor specificities for allele-specific HLA-A2.1 molecules(see, e.g., Falk et al., Nature 351:290-296, 1991; Hunt et al., Science255:1261-1263, 1992; Parker et al., J. Immunol. 149:3580-3587, 1992;Ruppert et al., Cell 74:929-937, 1993) and cross-reactive binding amongHLA-A2 and -A28 molecules have been described. (See, e.g., Fruci et al.,Human Immunol. 38:187-192, 1993; Tanigaki et al., Human Immunol.39:155-162, 1994; Del Guercio et al., J. Immunol. 154:685-693, 1995;Kast et al., J. Immunol. 152:3904-3912, 1994 for reviews of relevantdata). These primary anchor residues define the HLA-A2 supermotif; whichpresence in peptide ligands corresponds to the ability to bind severaldifferent HLA-A2 and -A28 molecules. The HLA-A2 supermotif comprisespeptide ligands with L, I, V, M, A, T, or Q as a primary anchor residueat position 2 and L, I, V, M, A, or T as a primary anchor residue at theC-terminal position of the epitope.

The corresponding family of HLA molecules (i.e., the HLA-A2 supertypethat binds these peptides) is comprised of at least: A*0201, A*0202,A*0203, A*0204, A*0205, A*0206, A*0207, A*0209, A*0214, A*6802, andA*6901. Other allele-specific HLA molecules predicted to be members ofthe A2 superfamily are shown in Table 5.

iii. HLA-A3 Supermotif

The HLA-A3 supermotif is characterized by the presence in peptideligands of A, L, I, V, M, S, or, T as a primary anchor at position 2,and a positively charged residue, R or K, at the C-terminal position ofthe epitope, e.g., in position 9 of 9-mers (see, e.g., Sidney et al.,Hum. Immunol. 45:79, 1996). Exemplary members of the correspondingfamily of HLA molecules (the HLA-A3 supertype) that bind the A3supermotif include at least A*0301, A*1101, A*3101, A*3301, and A*6801.Other allele-specific HLA molecules predicted to be members of the A3supertype are shown in Table 5.

iv. HLA-A24 Supermotif

The HLA-A24 supermotif is characterized by the presence in peptideligands of an aromatic (F, W, or Y) or hydrophobic aliphatic (L, I, V,M, or T) residue as a primary anchor in position 2, and Y, F, W, L, I,or M as primary anchor at the C-terminal position of the epitope (see,e.g., Sette and Sidney, Immunogenetics, in press, 1999). Thecorresponding family of HLA molecules that bind to the A24 supermotif(i.e., the A24 supertype) includes at least A*2402, A*3001, and A*2301.Other allele-specific HLA molecules predicted to be members of the A24supertype are shown in Table 5.

V. HLA-B7 Supermotif

The HLA-B7 supermotif is characterized by peptides bearing proline inposition 2 as a primary anchor, and a hydrophobic or aliphatic aminoacid (L, I, V, M, A, F, W, or Y) as the primary anchor at the C-terminalposition of the epitope. The corresponding family of HLA molecules thatbind the B7 supermotif (i.e., the HLA-B7 supertype) is comprised of atleast twenty six HLA-B proteins including: B*0702, B*0703, B*0704,B*0705, B*1508, B*3501, B*3502, B*3503, B*3504, B*3505, B*3506, B*3507,B*3508, B*5101, B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501,B*5502, B*5601, B*5602, B*6701, and B*7801 (see, e.g., Sidney, et al.,J. Immunol. 154:247, 1995; Barber, et al., Curr. Biol. 5:179, 1995;Hill, et al., Nature 360:434, 1992; Rammensee, et al., Immunogenetics41:178, 1995 for reviews of relevant data). Other allele-specific HLAmolecules predicted to be members of the B7 supertype are shown in Table5.

vi. HLA-B27 Supermotif

The HLA-B27 supermotif is characterized by the presence in peptideligands of a positively charged (R, H, or K) residue as a primary anchorat position 2, and a hydrophobic (F, Y, L, W, M, I, A, or V) residue asa primary anchor at the C-terminal position of the epitope (see, e.g.,Sidney and Sette, Immunogenetics, in press, 1999). Exemplary members ofthe corresponding family of HLA molecules that bind to the B27supermotif (i.e., the B27 supertype) include at least B*1401, B*1402,B*1509, B*2702, B*2703, B*2704, B*2705, B*2706, B*3801, B*3901, B*3902,and B*7301. Other allele-specific HLA molecules predicted to be membersof the B27 supertype are shown in Table 5.

vii. HLA-B44 Supermotif

The HLA-B44 supermotif is characterized by the presence in peptideligands of negatively charged (D or E) residues as a primary anchor inposition 2, and hydrophobic residues (F, W, Y, L, I, M, V, or A) as aprimary anchor at the C-terminal position of the epitope (see, e.g.,Sidney et al., Immunol. Today 17:261, 1996). Exemplary members of thecorresponding family of HLA molecules that bind to the B44 supermotif(i.e., the B44 supertype) include at least: B*1801, B*1802, B*3701,B*4001, B*4002, B*4006, B*4402, B*4403, and B*4006.

viii. HLA-B58 Supermotif

The HLA-B58 supermotif is characterized by the presence in peptideligands of a small aliphatic residue (A, S, or T) as a primary anchorresidue at position 2, and an aromatic or hydrophobic residue (F, W, Y,L, I, V, M, or A) as a primary anchor residue at the C-terminal positionof the epitope (see, e.g., Sidney and Sette, Immunogenetics, in press,1999 for reviews of relevant data). Exemplary members of thecorresponding family of HLA molecules that bind to the B58 supermotif(i.e., the B58 supertype) include at least: B*1516, B*1517, B*5701,B*5702, and B*5801. Other allele-specific HLA molecules predicted to bemembers of the B58 supertype are shown in Table 5.

ix. HLA-B62 Supermotif

The HLA-B62 supermotif is characterized by the presence in peptideligands of the polar aliphatic residue Q or a hydrophobic aliphaticresidue (L, V, M, I, or P) as a primary anchor in position 2, and ahydrophobic residue (F, W, Y, M, I, V, L, or A) as a primary anchor atthe C-terminal position of the epitope (see, e.g., Sidney and Sette,Immunogenetics, in press, 1999). Exemplary members of the correspondingfamily of HLA molecules that bind to the B62 supermotif (i.e., the B62supertype) include at least: B*1501, B*1502, B*1513, and B5201. Otherallele-specific HLA molecules predicted to be members of the B62supertype are shown in Table 5.

x. HLA-A1 Motif

The HLA-A1 motif is characterized by the presence in peptide ligands ofT, S, or M as a primary anchor residue at position 2 and the presence ofY as a primary anchor residue at the C-terminal position of the epitope.An alternative allele-specific A1 motif is characterized by a primaryanchor residue at position 3 rather than position 2. This motif ischaracterized by the presence of D, E, A, or S as a primary anchorresidue in position 3, and a Y as a primary anchor residue at theC-terminal position of the epitope (see, e.g., DiBrino et al., J.Immunol., 152:620, 1994; Kondo et al., Immunogenetics 45:249, 1997; andKubo et al., J. Immunol. 152:3913, 1994 for reviews of relevant data).

xi. HLA-A*0201 Motif

An HLA-A2*0201 motif was determined to be characterized by the presencein peptide ligands of L or M as a primary anchor residue in position 2,and L or V as a primary anchor residue at the C-terminal position of a9-residue peptide (see, e.g., Falk et al., Nature 351:290-296, 1991) andwas further found to comprise an I at position 2 and I or A at theC-terminal position of a nine amino acid peptide (see, e.g., Hunt etal., Science 255:1261-1263, Mar. 6, 1992; Parker et al., J. Immunol.149:3580-3587, 1992). The A*0201 allele-specific motif has also beendefined by the present inventors to additionally comprise V, A, T, or Qas a primary anchor residue at position 2, and M or T as a primaryanchor residue at the C-terminal position of the epitope (see, e.g.,Kast et al, J. Immunol. 152:3904-3912, 1994). Thus, the HLA-A*0201 motifcomprises peptide ligands with L, I, V, M, A, T, or Q as primary anchorresidues at position 2 and L, I, V, M, A, or T as a primary anchorresidue at the C-terminal position of the epitope. The preferred andtolerated residues that characterize the primary anchor positions of theHLA-A*0201 motif are identical to the residues describing the A2supermotif.

xii. HLA-A3 Motif

The HLA-A3 motif is characterized by the presence in peptide ligands ofL, M, V, I, S, A, T, F, C, G, or D as a primary anchor residue atposition 2, and the presence of K, Y, R, H, F, or A as a primary anchorresidue at the C-terminal position of the epitope (see, e.g., DiBrino etal., Proc. Natl. Acad. Sci. USA 90:1508, 1993; and Kubo et al., J.Immunol. 152:3913-3924, 1994).

xiii. HLA-A11 Motif

The HLA-A11 motif is characterized by the presence in peptide ligands ofV, T, M, L, I, S, A, G, N, C, D, or F as a primary anchor residue inposition 2, and K, R, Y, or H as a primary anchor residue at theC-terminal position of the epitope (see, e.g., Zhang et al., Proc. Natl.Acad. Sci. USA 90:2217-2221, 1993; and Kubo et al., J. Immunol.152:3913-3924, 1994).

xiv. HLA-A24 Motif

The HLA-A24 motif is characterized by the presence in peptide ligands ofY, F, W, or M as a primary anchor residue in position 2, and F, L, I, orW as a primary anchor residue at the C-terminal position of the epitope(see, e.g., Kondo et al., J. Immunol. 155:4307-4312, 1995; and Kubo etal., J. Immunol. 152:3913-3924, 1994).

5. Assays to Detect T-Cell Responses

Once heteroclitic analogs of the invention are synthesized, they can betested for the ability to elicit a T-cell response. The preparation andevaluation of motif-bearing peptides such as heteroclitic analogs aredescribed in PCT publications WO 94/20127 and WO 94/03205. Briefly,peptides comprising epitopes from a particular antigen are synthesizedand tested for their ability to bind to the appropriate HLA proteins.These assays may involve evaluating the binding of a peptide of theinvention to purified HLA Class I molecules in relation to the bindingof a radioiodinated reference peptide. Alternatively, cells expressingempty Class I molecules (i.e. lacking peptide therein) may be evaluatedfor peptide binding by immunofluorescent staining and flowmicrofluorimetry. Other assays that may be used to evaluate peptidebinding include peptide-dependent Class I assembly assays and/or theinhibition of CTL recognition by peptide competition. Those peptidesthat bind to the Class I molecule, typically with an affinity of 500 nMor less, are further evaluated for their ability to serve as targets forCTLs derived from infected or immunized individuals, as well as fortheir capacity to induce primary in vitro or in vivo CTL responses thatcan give rise to CTL populations capable of reacting with selectedtarget cells associated with a disease.

Conventional assays utilized to detect T cell responses includeproliferation assays, lymphokine secretion assays, direct cytotoxicityassays, and limiting dilution assays. Such assays are useful incomparing the induction of immune responses by heteroclitic analogpeptides to response induced by non-heteroclitic analogs Class Ipeptides (e.g., from which the heterocloitic analog sequence was based).For example, antigen-presenting cells that have been incubated with apeptide can be assayed for the ability to induce CTL responses inresponder cell populations. Antigen-presenting cells can be normal cellssuch as peripheral blood mononuclear cells or dendritic cells.Alternatively, mutant non-human mammalian cell lines that are deficientin their ability to load Class I molecules with internally processedpeptides and that have been transfected with the appropriate human ClassI gene, may be used to test for the capacity of the peptide to induce invitro primary CTL responses.

Peripheral blood mononuclear cells (PBMCs) may be used as the respondercell source of CTL precursors. The appropriate antigen-presenting cellsare incubated with peptide, after which the peptide-loadedantigen-presenting cells are then incubated with the responder cellpopulation under optimized culture conditions. Positive CTL activationcan be determined by assaying the culture for the presence of CTLs thatkill radio-labeled target cells, both specific peptide-pulsed targets aswell as target cells expressing endogenously processed forms of theantigen from which the peptide sequence was derived.

Additionally, a method has been devised which allows directquantification of antigen-specific T cells by staining withFluorescein-labelled HLA tetrameric complexes (Altman, J. D. et al.,Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman, J. D. et al., Science274:94, 1996). Other relatively recent technical developments includestaining for intracellular lymphokines, and interferon-γ release assaysor Elispot assays. Tetramer staining, intracellular lymphokine stainingand Elispot assays all appear to be at least 10-fold more sensitive thanmore conventional assays (Lalvani, A. et al., J. Exp. Med. 186:859,1997; Dunbar, P. R. et al., Curr. Biol. 8:413, 1998; Murali-Krishna, K.et al., Immunity 8:177, 1998).

If desired, HTL activation may also be assessed using such techniquesknown to those in the art such as T cell proliferation and secretion oflymphokines, e.g. IL-2 (see, e.g. Alexander, et al., Immunity 1:751-761,1994).

Alternatively, immunization of HLA transgenic mice can be used todetermine immunogenicity of peptide epitopes. Several transgenic mousemodels including mice with human A2.1, A11 (which can additionally beused to analyze HLA-A3 epitopes), and B7 alleles have been characterizedand others (e.g., transgenic mice for HLA-A1 and A24) are beingdeveloped. HLA-DR1 and HLA-DR3 mouse models have also been developed.Additional transgenic mouse models with other HLA alleles may begenerated as necessary. The mice may be immunized with peptidesemulsified in Incomplete Freund's Adjuvant and the resulting T cellstested for their capacity to recognize peptide-pulsed target cells andtarget cells transfected with appropriate genes. CTL responses may beanalyzed using cytotoxicity assays described above. Similarly, HTLresponses may be analyzed using such assays as T cell proliferation orsecretion of lymphokines.

Heteroclitic analogs of the invention often induce both Th1 and Th2cytokine responses. Therefore, one method to compare a heterocliticcandidate with a preselected Class I peptide is to test the induction ofTh1 and Th2 cytokines. The preselected Class I peptide will typically bea peptide from which the heteroclitic analog is derived, or if such apeptide does not exist, a Class I peptide with the highest similarity tothe candidate. Heteroclitic analogs of the invention typically induceboth Th1 and Th2 cytokine responses, but at a level greatly enhancedcompared to the Class I peptide from which the analog was derived. Forexample, a given heteroclitic analog will stimulate an equivalent levelof Th1 or Th2 cytokine (50 to 100 pg/ml) at a 10-fold or lower dosecompared to the wildtype peptide from which the analog was derived.Additionally, where the Class I peptide induces only, or mainly, eithera Th1 or Th2 response, a heteroclitic analog may induce both Th1 and Th2responses. Th1 cytokines include, e.g., IFNγ, IL-2 and IL-3. Th2cytokines include, e.g., IL-4, IL-5, IL-6 and IL-10. Production ofcytokines can be measured, for example, using ELISA or otherimmunological quantitation methods. See, e.g., McKinney, et al. Journalof Immunological Methods 237:105-117 (2000).

6. Use of Peptide Epitopes as Diagnostic Agents and for EvaluatingImmune Responses

In one embodiment of the invention, heteroclitic analog peptides asdescribed herein are used as reagents to evaluate an immune response.The immune response to be evaluated is induced by using as an immunogenany agent that may result in the induction of antigen-specific CTLs orHTLs that recognize and bind to the peptide epitope(s) to be employed asthe reagent. The peptide reagent need not be used as the immunogen.Assay systems that are used for such an analysis include relativelyrecent technical developments such as tetramers, staining forintracellular lymphokines and interferon release assays, or Elispotassays.

For example, peptides of the invention are used in tetramer stainingassays to assess peripheral blood mononuclear cells for the presence ofantigen-specific CTLs following exposure to a tumor cell antigen or animmunogen. The HLA-tetrameric complex is used to directly visualizeantigen-specific CTLs (see, e.g., Ogg et al., Science 279:2103-2106,1998; and Altman et al., Science 174:94-96, 1996) and determine thefrequency of the antigen-specific CTL population in a sample ofperipheral blood mononuclear cells. A tetramer reagent using a peptideof the invention is generated as follows: A peptide that binds to an HLAmolecule is refolded in the presence of the corresponding HLA heavychain and β₂-microglobulin to generate a trimolecular complex. Thecomplex is biotinylated at the carboxyl terminal end of the heavy chainat a site that was previously engineered into the protein. Tetramerformation is then induced by the addition of streptavidin. By means offluorescently labeled streptavidin, the tetramer can be used to stainantigen-specific cells. The cells can then be identified, for example,by flow cytometry. Such an analysis may be used for diagnostic orprognostic purposes. Cells identified by the procedure can also be usedfor therapeutic purposes.

Peptides of the invention are also used as reagents to evaluate immunerecall responses (see, e.g., Bertoni, et al., J. Clin. Invest.100:503-513, 1997 and Penna, et al., J. Exp. Med. 174:1565-1570, 1991).For example, patient PBMC samples from individuals with cancer areanalyzed for the presence of antigen-specific CTLs or HTLs usingspecific peptides. A blood sample containing mononuclear cells can beevaluated by cultivating the PBMCs and stimulating the cells with apeptide of the invention. After an appropriate cultivation period, theexpanded cell population can be analyzed, for example, for CTL or forHTL activity.

The peptides are also used as reagents to evaluate the efficacy of avaccine. PBMCs obtained from a patient vaccinated with an immunogen areanalyzed using, for example, either of the methods described above. Thepatient is HLA typed, and peptide epitope reagents that recognize theallele-specific molecules present in that patient are selected for theanalysis. The immunogenicity of the vaccine is indicated by the presenceof epitope-specific CTLs and/or HTLs in the PBMC sample.

The peptides of the invention are also used to make antibodies, usingtechniques well known in the art (see, e.g. CURRENT PROTOCOLS INIMMUNOLOGY, Wiley/Greene, NY; and Antibodies A Laboratory Manual, Harlowand Lane, Cold Spring Harbor Laboratory Press, 1989), which may beuseful as reagents to diagnose or monitor cancer. Such antibodiesinclude those that recognize a peptide in the context of an HLAmolecule, i.e., antibodies that bind to a peptide-MHC complex.

7. Vaccine Compositions

Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more peptides as describedherein are further embodiments of the invention. Once appropriatelyimmunogenic epitopes have been defined, they can be sorted and deliveredby various means, herein referred to as “vaccine” compositions. Suchvaccine compositions can include, for example, lipopeptides (e.g.,Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptidecompositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”)microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294,1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine13:675-681, 1995), peptide compositions contained in immune stimulatingcomplexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875,1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigenpeptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci.USA. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32,1996), peptides formulated as multivalent peptides; peptides for use inballistic delivery systems, typically crystallized peptides, viraldelivery vectors (Perkus, M. E. et al., In: Concepts in vaccinedevelopment, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. etal., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986;Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al.,J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535,1990), particles of viral or synthetic origin (e.g., Kofler, N. et al.,J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol.30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995),adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev.Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993),liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L.,Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA(Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L.A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In:Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996;Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 andEldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeteddelivery technologies, also known as receptor mediated targeting, suchas those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also beused.

Vaccines of the invention include nucleic acid-mediated modalities. DNAor RNA encoding one or more of the peptides of the invention can also beadministered to a patient. This approach is described, for instance, inWolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos.5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO98/04720; and in more detail below. Examples of DNA-based deliverytechnologies include “naked DNA”, facilitated (bupivicaine, polymers,peptide-mediated) delivery, cationic lipid complexes, andparticle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g.,U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, the peptides ofthe invention can also be expressed by viral or bacterial vectors.Examples of expression vectors include attenuated viral hosts, such asvaccinia or fowlpox. As an example of this approach, vaccinia virus isused as a vector to express nucleotide sequences that encode thepeptides of the invention. Upon introduction into a host bearing atumor, the recombinant vaccinia virus expresses the immunogenic peptide,and thereby elicits a host CTL and/or HTL response. Vaccinia vectors andmethods useful in immunization protocols are described in, e.g., U.S.Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCGvectors are described in Stover et al., Nature 351:456-460 (1991). Awide variety of other vectors useful for therapeutic administration orimmunization of the peptides of the invention, e.g. adeno andadeno-associated virus vectors, retroviral vectors, Salmonella typhivectors, detoxified anthrax toxin vectors, and the like, will beapparent to those skilled in the art from the description herein.

Furthermore, vaccines in accordance with the invention encompasscompositions of one or more of the claimed peptides. A peptide can bepresent in a vaccine individually. Alternatively, the peptide can existas a homopolymer comprising multiple copies of the same peptide, or as aheteropolymer of various peptides. Polymers have the advantage ofincreased immunological reaction and, where different peptide epitopesare used to make up the polymer, the additional ability to induceantibodies and/or CTLs that react with different antigenic determinantsof the pathogenic organism or tumor-related peptide targeted for animmune response. The composition can be a naturally occurring region ofan antigen or can be prepared, e.g., recombinantly or by chemicalsynthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly L-lysine,poly L-glutamic acid, influenza, hepatitis B virus core protein, and thelike. The vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, preferably phosphatebuffered saline. The vaccines also typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are examples of materials well known in theart. Additionally, as disclosed herein, CTL responses can be primed byconjugating peptides of the invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS).

Upon immunization with a peptide composition in accordance with theinvention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later infection, or at leastpartially resistant to developing an ongoing chronic infection, orderives at least some therapeutic benefit when the antigen wastumor-associated.

In some embodiments, it may be desirable to combine the heterocliticanalog peptides of the invention with components that induce orfacilitate neutralizing antibody and or helper T cell responses to thetarget antigen of interest. A preferred embodiment of such a compositioncomprises Class I and Class II epitopes in accordance with theinvention. An alternative embodiment of such a composition comprises aClass I and/or Class II epitope in accordance with the invention, alongwith a pan-DR binding peptide such as PADRE™ (Epimmune, San Diego,Calif.) molecule (described, for example, in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells(APC), such as dendritic cells (DC), as a vehicle to present peptides ofthe invention. Vaccine compositions can be created in vitro, followingdendritic cell mobilization and harvesting, whereby loading of dendriticcells occurs in vitro. For example, dendritic cells are transfected,e.g., with a minigene in accordance with the invention, or are pulsedwith peptides. The dendritic cell can then be administered to a patientto elicit immune responses in vivo.

Vaccine compositions, either DNA- or peptide-based, can also beadministered in vivo in combination with dendritic cell mobilizationwhereby loading of dendritic cells occurs in vivo.

Antigenic peptides are used to elicit a CTL and/or HTL response ex vivo,as well. The resulting CTL or HTL cells, can be used to treat tumors inpatients that do not respond to other conventional forms of therapy, orwill not respond to a therapeutic vaccine peptide or nucleic acid inaccordance with the invention. Ex vivo CTL or HTL responses to aparticular tumor-associated antigen are induced by incubating in tissueculture the patient's, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells, such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (aninfected cell or a tumor cell). Transfected dendritic cells may also beused as antigen presenting cells.

The vaccine compositions of the invention can also be used incombination with other treatments used for cancer, including use incombination with immune adjuvants such as IL-2, IL-12, GM-CSF, and thelike.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition for use ina vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles are balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

1) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with tumor clearance.For HLA Class I, this includes 3-4 epitopes that come from at least onetumor-associated antigen (TAA). For HLA Class II, a similar rationale isemployed; again 3-4 epitopes are selected from at least one TAA (seee.g., Rosenberg et al, Science 278:1447-1450). Epitopes from one TAA maybe used in combination with epitopes from one or more additional TAAs toproduce a vaccine that targets tumors with varying expression patternsof frequently-expressed TAAs.

2) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, often 200 nM or less; and for Class II an IC₅₀of 1000 nM or less.

3) Sufficient supermotif bearing-peptides, or a sufficient array ofallele-specific motif-bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess the breadth, or redundancyof, population coverage.

4) When selecting epitopes from cancer-related antigens it is oftenuseful to select analogs because the patient may have developedtolerance to the native epitope. When selecting epitopes for infectiousdisease-related antigens, it is preferable to select either native oranaloged epitopes.

5) Of particular relevance are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A nested peptide sequence can comprise bothHLA Class I and HLA Class II epitopes. When providing nested epitopes, ageneral objective is to provide the greatest number of epitopes persequence. Thus, an aspect is to avoid providing a peptide that is anylonger than the amino terminus of the amino terminal epitope and thecarboxyl terminus of the carboxyl terminal epitope in the peptide. Whenproviding a multi-epitopic sequence, such as a sequence comprisingnested epitopes, it is generally important to screen the sequence inorder to insure that it does not have pathological or other deleteriousbiological properties.

6) If a polyepitopic protein is created, or when creating a minigene, anobjective is to generate the smallest peptide that encompasses theepitopes of interest. This principle is similar, if not the same as thatemployed when selecting a peptide comprising nested epitopes. However,with an artificial polyepitopic peptide, the size minimization objectiveis balanced against the need to integrate any spacer sequences betweenepitopes in the polyepitopic protein. Spacer amino acid residues can,for example, be introduced to avoid junctional epitopes (an epitoperecognized by the immune system, not present in the target antigen, andonly created by the man-made juxtaposition of epitopes), or tofacilitate cleavage between epitopes and thereby enhance epitopepresentation. Junctional epitopes are generally to be avoided becausethe recipient may generate an immune response to that non-nativeepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

8. Minigene Vaccines

A number of different approaches are available which allow simultaneousdelivery of multiple epitopes. Nucleic acids encoding the peptides ofthe invention are a particularly useful embodiment of the invention.Epitopes for inclusion in a minigene are preferably selected accordingto the guidelines set forth in the previous section. A preferred meansof administering nucleic acids encoding the peptides of the inventionuses minigene constructs encoding a peptide comprising one or multipleepitopes of the invention.

The use of multi-epitope minigenes is described below and in, e.g.,co-pending application U.S. Ser. No. 09/311,784; Ishioka et al., J.Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol.71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996;Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine16:426, 1998. For example, a multi-epitope DNA plasmid encodingsupermotif- and/or motif-bearing epitopes (e.g., PSA, PSM, PAP, and hK2)derived from multiple regions of a TAA, a pan_DR binding peptide such asthe PADRE™ universal helper T cell epitope, and an endoplasmicreticulum-translocating signal sequence can be engineered. A vaccine mayalso comprise epitopes that are derived from other TAAs.

The immunogenicity of a multi-epitopic minigene can be tested intransgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA Class I epitopes, HLA Class II epitopes, a ubiquitinationsignal sequence, and/or an endoplasmic reticulum targeting signal. Inaddition, HLA presentation of CTL and HTL epitopes may be improved byincluding synthetic (e.g. poly-alanine) or naturally-occurring flankingsequences adjacent to the CTL or HTL epitopes; these larger peptidescomprising the epitope(s) are within the scope of the invention.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, orfor HTL responses, pan-DR binding proteins (e.g., PADRE™, Epimmune, SanDiego, Calif.). Helper (HTL) epitopes can be joined to intracellulartargeting signals and expressed separately from expressed CTL epitopes;this allows direction of the HTL epitopes to a cell compartmentdifferent than that of the CTL epitopes. If required, this couldfacilitate more efficient entry of HTL epitopes into the HLA Class IIpathway, thereby improving HTL induction. In contrast to HTL or CTLinduction, specifically decreasing the immune response by co-expressionof immunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffered saline (PBS). This approach, known as“naked DNA,” is currently being used for intramuscular (IM)administration in clinical trials. To maximize the immunotherapeuticeffects of minigene DNA vaccines, an alternative method for formulatingpurified plasmid DNA may be desirable. A variety of methods have beendescribed, and new techniques may become available. Cationic lipids,glycolipids, and fusogenic liposomes can also be used in the formulation(see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite,BioTechniques 6 (7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309;and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). Inaddition, peptides and compounds referred to collectively as protective,interactive, non-condensing compounds (PINC) could also be complexed topurified plasmid DNA to influence variables such as stability,intramuscular dispersion, or trafficking to specific organs or celltypes.

Target cell sensitization can be used as a functional assay forexpression and HLA Class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration can be formulation dependent (e.g., IM for DNA in PBS,intraperitoneal (IP) for lipid-complexed DNA). Twenty-one days afterimmunization, splenocytes are harvested and restimulated for one week inthe presence of peptides encoding each epitope being tested. Thereafter,for CTL effector cells, assays are conducted for cytolysis ofpeptide-loaded, ⁵¹Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs. Immunogenicity of HTLepitopes is evaluated in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered intradermally, e.g.by injection or ballistic delivery as described, for instance, in U.S.Pat. No. 5,204,253. Using this technique, particles comprised solely ofDNA are administered. In a further alternative embodiment, DNA can beadhered to particles, such as gold particles.

Minigenes can also be delivered using other bacterial or viral deliverysystems well known in the art, e.g., an expression construct encodingepitopes of the invention can be incorporated into a viral vector suchas vaccinia.

9. Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising the peptides of the present inventioncan be modified to provide desired attributes, such as improved serumhalf-life, or to enhance immunogenicity.

For instance, the ability of a peptide to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response. Theuse of T helper epitopes in conjunction with CTL epitopes to enhanceimmunogenicity is illustrated, for example, in the co-pendingapplications U.S. Ser. No. 08/820,360, U.S. Ser. No. 08/197,484, andU.S. Ser. No. 08/464,234.

Although a CTL peptide can be directly linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in the majority of the population. This can beaccomplished by selecting amino acid sequences that bind to many, most,or all of the HLA Class II molecules. These are known as “looselyHLA-restricted” or “promiscuous” T helper sequences. Examples ofpeptides that are promiscuous include sequences from antigens such astetanus toxoid at positions 830-843 (QYIKANSKFIGITE) (SEQ ID NO:32),Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398(DIEKKIAKMEKASSVFNVVNS) (SEQ ID NO:33), and Streptococcus 18 kD proteinat positions 116 (GAVDSILGGVATYGAA) (SEQ ID NO:34). Other examplesinclude peptides bearing a DR 1-4-7 supermotif, or either of the DR3motifs.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed to most preferrably bind most HLA-DR (human HLAClass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: aKXVAAWTLKAAa, where “X” is either cyclohexylalanine(SEQ ID NO: 35), phenylalanine (SEQ ID NO:36), or tyrosine (SEQ IDNO:37), and “a” is either D-alanine or L-alanine, has been found to bindto most HLA-DR alleles, and to stimulate the response of T helperlymphocytes from most individuals, regardless of their HLA type. Analternative of a pan-DR binding epitope comprises all “L” natural aminoacids and can be provided in the form of nucleic acids that encode theepitope.

HTL peptide epitopes can also be modified to alter their biologicalproperties. For example, they can be modified to include D-amino acidsto increase their resistance to proteases and thus extend their serumhalf life, or they can be conjugated to other molecules such as lipids,proteins, carbohydrates, and the like to increase their biologicalactivity. For example, a T helper peptide can be conjugated to one ormore palmitic acid chains at either the amino or carboxyl termini.

10. Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primescytotoxic T lymphocytes. Lipids have been identified as agents capableof priming CTL in vivo against viral antigens. For example, palmiticacid residues can be attached to the ε- and α-amino groups of a lysineresidue and then linked, e.g., via one or more linking residues such asGly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. Thelipidated peptide can then be administered either directly in a micelleor particle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. A preferred immunogenic compositioncomprises palmitic acid attached to ε- and α-amino groups of Lys, whichis attached via linkage, e.g., Ser-Ser, to the amino terminus of theimmunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P₃CSS, for example,and the lipopeptide administered to an individual to specifically primea CTL response to the target antigen. Moreover, because the induction ofneutralizing antibodies can also be primed with P₃CSS-conjugatedepitopes, two such compositions can be combined to more effectivelyelicit both humoral and cell-mediated responses.

CTL and/or HTL peptides can also be modified by the addition of aminoacids to the termini of a peptide to provide for ease of linkingpeptides one to another, for coupling to a carrier support or largerpeptide, for modifying the physical or chemical properties of thepeptide or oligopeptide, or the like. Amino acids such as tyrosine,cysteine, lysine, glutamic or aspartic acid, or the like, can beintroduced at the C- or N-terminus of the peptide or oligopeptide,particularly Class I peptides. However, it is to be noted thatmodification at the carboxyl terminus of a CTL epitope may, in somecases, alter binding characteristics of the peptide. In addition, thepeptide or oligopeptide sequences can differ from the natural sequenceby being modified by terminal-NH₂ acylation, e.g., by alkanoyl (C₁-C₂₀)or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia,methylamine, etc. In some instances these modifications may providesites for linking to a support or other molecule.

11. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTLPeptides

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood. Apharmaceutical to facilitate harvesting of DC can be used, such asProgenipoietin™ (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsingthe DC with peptides and prior to reinfusion into patients, the DC arewashed to remove unbound peptides. In this embodiment, a vaccinecomprises peptide-pulsed DCs that present the pulsed peptide epitopescomplexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of whichstimulate CTL response to one or more antigens of interest. Optionally,a helper T cell peptide such as a PADRE™ family molecule, can beincluded to facilitate the CTL response.

12. Administration of Vaccines for Therapeutic or Prophylactic Purposes

The peptides of the present invention and pharmaceutical and vaccinecompositions of the invention are typically used therapeutically totreat cancer. Vaccine compositions containing the peptides of theinvention are typically administered to a cancer patient who has amalignancy associated with expression of one or more antigens.Alternatively, vaccine compositions can be administered to an individualsusceptible to, or otherwise at risk for developing cancer.

In therapeutic applications, peptide and/or nucleic acid compositionsare administered to a patient in an amount sufficient to elicit aneffective CTL and/or HTL response to the tumor antigen and to cure or atleast partially arrest or slow symptoms and/or complications. An amountadequate to accomplish this is defined as “therapeutically effectivedose.” Amounts effective for this use will depend on, e.g., theparticular composition administered, the manner of administration, thestage and severity of the disease being treated, the weight and generalstate of health of the patient, and the judgment of the prescribingphysician.

As noted above, peptides comprising CTL and/or HTL epitopes of theinvention induce immune responses when presented by HLA molecules andcontacted with a CTL or HTL specific for an epitope comprised by thepeptide. The peptides (or DNA encoding them) can be administeredindividually or as fusions of one or more peptide sequences. The mannerin which the peptide is contacted with the CTL or HTL is not critical tothe invention. For instance, the peptide can be contacted with the CTLor HTL either in vivo or in vitro. If the contacting occurs in vivo, thepeptide itself can be administered to the patient, or other vehicles,e.g., DNA vectors encoding one or more peptides, viral vectors encodingthe peptide(s), liposomes and the like, can be used, as describedherein.

When the peptide is contacted in vitro, the vaccinating agent cancomprise a population of cells, e.g., peptide-pulsed dendritic cells, orTAA-specific CTLs, which have been induced by pulsing antigen-presentingcells in vitro with the peptide or by transfecting antigen-presentingcells with a minigene of the invention. Such a cell population issubsequently administered to a patient in a therapeutically effectivedose.

For therapeutic use, administration should generally begin at the firstdiagnosis of cancer. This is followed by boosting doses until at leastsymptoms are substantially abated and for a period thereafter. Theembodiment of the vaccine composition (i.e., including, but not limitedto embodiments such as peptide cocktails, polyepitopic polypeptides,minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered tothe patient may vary according to the stage of the disease or thepatient's health status. For example, a vaccine comprising TAA-specificCTLs may be more efficacious in killing tumor cells in patients withadvanced disease than alternative embodiments.

The vaccine compositions of the invention may also be usedtherapeutically in combination with treatments such as surgery. Anexample is a situation in which a patient has undergone surgery toremove a primary tumor and the vaccine is then used to slow or preventrecurrence and/or metastasis.

Where susceptible individuals, e.g., individuals who may be diagnosed asbeing genetically pre-disposed to developing a prostate tumor, areidentified prior to diagnosis of cancer, the composition can be targetedto them, thus minimizing the need for administration to a largerpopulation.

The dosage for an initial therapeutic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1,000μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.Dosage values for a human typically range from about 500 μg to about50,000 μg per 70 kilogram patient. Initial doses followed by boostingdoses at established intervals, e.g., from four weeks to six months, maybe required, possibly for a prolonged period of time to effectivelytreat a patient. Boosting dosages of between about 1.0 μg to about50,000 μg of peptide pursuant to a boosting regimen over weeks to monthsmay be administered depending upon the patient's response and conditionas determined by measuring the specific activity of CTL and HTL obtainedfrom the patient's blood.

Administration should continue until at least clinical symptoms orlaboratory tests indicate that the tumor has been eliminated or that thetumor cell burden has been substantially reduced and for a periodthereafter. The dosages, routes of administration, and dose schedulesare adjusted in accordance with methodologies known in the art.

In certain embodiments, peptides and compositions of the presentinvention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used asprophylactic agents. For example, the compositions can be administeredto individuals at risk of developing prostate cancer. Generally thedosage for an initial prophylactic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1000μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.Dosage values for a human typically range from about 500 μg to about50,000 μg per 70 kilogram patient. This is followed by boosting dosagesof between about 1.0 μg to about 50,000 μg of peptide administered atdefined intervals from about four weeks to six months after the initialadministration of vaccine. The immunogenicity of the vaccine may beassessed by measuring the specific activity of CTL and HTL obtained froma sample of the patient's blood.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, intrathecal, or local administration.Preferably, the pharmaceutical compositions are administered parentally,e.g., intravenously, subcutaneously, intradermally, or intramuscularly.Thus, the invention provides compositions for parenteral administrationwhich comprise a solution of the immunogenic peptides dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH-adjusting and buffering agents, tonicityadjusting agents, wetting agents, preservatives, and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

A human unit dose form of the peptide composition is typically includedin a pharmaceutical composition that comprises a human unit dose of anacceptable carrier, preferably an aqueous carrier, and is administeredin a volume of fluid that is known by those of skill in the art to beused for administration of such compositions to humans (see, e.g.,Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro,Editor, Mack Publishing Co., Easton, Pa., 1985).

The peptides of the invention may also be administered via liposomes,which serve to target the peptides to a particular tissue, such aslymphoid tissue, or to target selectively to infected cells, as well asto increase the half-life of the peptide composition. Liposomes includeemulsions, foams, micelles, insoluble monolayers, liquid crystals,phospholipid dispersions, lamellar layers and the like. In thesepreparations, the peptide to be delivered is incorporated as part of aliposome, alone or in conjunction with a molecule which binds to areceptor prevalent among lymphoid cells, such as monoclonal antibodieswhich bind to the CD45 antigen, or with other therapeutic or immunogeniccompositions. Thus, liposomes either filled or decorated with a desiredpeptide of the invention can be directed to the site of lymphoid cells,where the liposomes then deliver the peptide compositions. Liposomes foruse in accordance with the invention are formed from standardvesicle-forming lipids, which generally include neutral and negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of, e.g., liposome size,acid lability and stability of the liposomes in the blood stream. Avariety of methods are available for preparing liposomes, as describedin, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), andU.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc., in a dose which variesaccording to, inter alia, the manner of administration, the peptidebeing delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, the immunogenic peptides are preferablysupplied in finely divided form along with a surfactant and propellant.Typical percentages of peptides are 0.01%-20% by weight, preferably1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from 6 to 22 carbon atoms,such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute 0.1%-20% byweight of the composition, preferably 0.25-5%. The balance of thecomposition is ordinarily propellant. A carrier can also be included, asdesired, as with, e.g., lecithin for intranasal delivery.

13. Kits

The peptide and nucleic acid compositions of this invention can beprovided in kit form together with instructions for vaccineadministration. Typically the kit would include desired peptidecompositions in a container, preferably in unit dosage form andinstructions for administration. An alternative kit would include aminigene construct with desired nucleic acids of the invention in acontainer, preferably in unit dosage form together with instructions foradministration. Lymphokines such as IL-2 or IL-12 may also be includedin the kit. Other kit components that may also be desirable include, forexample, a sterile syringe, booster dosages, and other desiredexcipients.

Epitopes in accordance with the present invention were successfully usedto induce an immune response. Immune responses with these epitopes havebeen induced by administering the epitopes in various forms. Theepitopes have been administered as peptides, as nucleic acids, and asviral vectors comprising nucleic acids that encode the epitope(s) of theinvention. Upon administration of peptide-based epitope forms, immuneresponses have been induced by direct loading of an epitope onto anempty HLA molecule that is expressed on a cell, and via internalizationof the epitope and processing via the HLA Class I pathway; in eitherevent, the HLA molecule expressing the epitope was then able to interactwith and induce a CTL response. Peptides can be delivered directly orusing such agents as liposomes. They can additionally be delivered usingballistic delivery, in which the peptides are typically in a crystallineform. When DNA is used to induce an immune response, it is administeredeither as naked DNA, generally in a dose range of approximately 1-5 mg,or via the ballistic “gene gun” delivery, typically in a dose range ofapproximately 10-100 μg. The DNA can be delivered in a variety ofconformations, e.g., linear, circular etc. Various viral vectors havealso successfully been used that comprise nucleic acids which encodeepitopes in accordance with the invention.

Accordingly compositions in accordance with the invention exist inseveral forms. Embodiments of each of these composition forms inaccordance with the invention have been successfully used to induce animmune response.

One composition in accordance with the invention comprises a pluralityof peptides. This plurality or cocktail of peptides is generally admixedwith one or more pharmaceutically acceptable excipients. The peptidecocktail can comprise multiple copies of the same peptide or cancomprise a mixture of peptides. The peptides can be analogs of naturallyoccurring epitopes. The peptides can comprise artificial amino acidsand/or chemical modifications such as addition of a surface activemolecule, e.g., lipidation; acetylation, glycosylation, biotinylation,phosphorylation etc. The peptides can be CTL or HTL epitopes. In apreferred embodiment the peptide cocktail comprises a plurality ofdifferent CTL epitopes and at least one HTL epitope. The HTL epitope canbe naturally or non-naturally (e.g., PADRE®, Epimmune Inc., San Diego,Calif.). The number of distinct epitopes in an embodiment of theinvention is generally a whole unit integer from one through one hundredfifty (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, . . . , 150).

An additional embodiment of a composition in accordance with theinvention comprises a polypeptide multi-epitope construct, i.e., apolyepitopic peptide. Polyepitopic peptides in accordance with theinvention are prepared by use of technologies well-known in the art. Byuse of these known technologies, epitopes in accordance with theinvention are connected one to another. The polyepitopic peptides can belinear or non-linear, e.g., multivalent. These polyepitopic constructscan comprise artificial amino acids, spacing or spacer amino acids,flanking amino acids, or chemical modifications between adjacent epitopeunits. The polyepitopic construct can be a heteropolymer or ahomopolymer. The polyepitopic constructs generally comprise epitopes ina quantity of any whole unit integer between 2-1.50 (e.g., 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, . . . , 150). The polyepitopic construct can comprise CTLand/or HTL epitopes. One or more of the epitopes in the construct can bemodified, e.g., by addition of a surface active material, e.g. a lipid,or chemically modified, e.g., acetylation, etc. Moreover, bonds in themultiepitopic construct can be other than peptide bonds, e.g., covalentbonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionicbonds, etc.

Alternatively, a composition in accordance with the invention comprisesconstruct which comprises a series, sequence, stretch, etc., of aminoacids that have homology to (i.e., corresponds to or is contiguous with)to a native sequence. This stretch of amino acids comprises at least onesubsequence of amino acids that, if cleaved or isolated from the longerseries of amino acids, functions as an HLA Class I or HLA Class IIepitope in accordance with the invention. In this embodiment, thepeptide sequence is modified, so as to become a construct as definedherein, by use of any number of techniques known or to be provided inthe art. The polyepitopic constructs can contain homology to a nativesequence in any whole unit integer increment from 70-100% (e.g., 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or, 100 percent).

A further embodiment of a composition in accordance with the inventionis an antigen presenting cell that comprises one or more epitopes inaccordance with the invention. The antigen presenting cell can be a“professional” antigen presenting cell, such as a dendritic cell. Theantigen presenting cell can comprise the epitope of the invention by anymeans known or to be determined in the art. Such means include pulsingof dendritic cells with one or more individual epitopes or with one ormore peptides that comprise multiple epitopes, by nucleic acidadministration such as ballistic nucleic acid delivery or by othertechniques in the art for administration of nucleic acids, includingvector-based, e.g. viral vector, delivery of nucleic acids.

Further embodiments of compositions in accordance with the inventioncomprise nucleic acids that encode one or more peptides of theinvention, or nucleic acids which encode a polyepitopic peptide inaccordance with the invention. As appreciated by one of ordinary skillin the art, various nucleic acids compositions will encode the samepeptide due to the redundancy of the genetic code. Each of these nucleicacid compositions falls within the scope of the present invention. Thisembodiment of the invention comprises DNA or RNA, and in certainembodiments a combination of DNA and RNA. It is to be appreciated thatany composition comprising nucleic acids that will encode a peptide inaccordance with the invention or any other peptide based composition inaccordance with the invention, falls within the scope of this invention.

EXAMPLES Preparation A Peptide Synthesis and Generation of PeptideAnalogs

The peptides used in these examples are shown in Table 1. All of thewildtype human CTL epitopes derived from tumor-associated antigens, aswell as the wildtype viral epitopes derived from the polymerase genes ofthe HIV and hepatitis B virus (HBV²), have shown immunogenicity in humanand transgenic mouse systems (Kawashima, I., et al., Human Immunol.(1998) 59:1; Ishioka, G., et al., J. Immunol. (1999) 162:3915; Epimmune,unpublished data).

Peptides that were tested initially for heteroclitic activity weresynthesized by Chiron Technologies (Victor, Australia). Peptidesrequiring further biological characterization were synthesized atEpimmune using conventional methods (Ruppert, J., et al., Cell (1993)74:929) and their purity was routinely >95%, as determined by analyticalreverse-phase HPLC. The identity of the latter peptides was confirmed bymass spectral analysis.

Preparation B Scheme for Selection of Single Amino Acid Substitutions

Table 2 shows the similarity assignments between any given amino acidpair so that a given amino acid substitution could be characterized asbeing a conservative, semi-conservative, or non-conservativesubstitution.

The degree of similarity between amino acid pairs was quantified byaveraging, for each amino acid pair, the rank coefficient scores forPAM250, hydrophobicity, and side chain volume as described below. Basedon the average values of these composite rankings, the table shows eachpair to be conserved, semi-conserved or non-conserved.

The Dayhoff PAM250 score (Dayhoff, M. O., et al., Atlas of ProteinSequence and Structure, Vol. 5, suppl. 3. (1978) M. O. Dayhoff, ed.National Biomedical Research Foundation, Washington D.C., p. 345;Creighton, T. E., Proteins: structures and molecular properties (1993)(2nd edition) W.H. Freeman and Company, NY;http://prowl.rockefeller.edu/aainfo/pam250. html) is a commonly utilizedprotein alignment scoring matrix which measures the percentage ofacceptable point mutations (PAM) within a defined time frame. Thefrequencies of these mutations are different from what would be expectedfrom the probability of random mutations, and presumably reflect a biasdue to the degree of physical and chemical similarity of the amino acidpair involved in the substitution. To obtain a score of amino acidsimilarity that could be standardized with other measures of similarity,the PAM250 scores were converted to a rank value, where I indicates thehighest probability of being an accepted mutation.

The most commonly utilized scales to represent the relativehydrophobicity of the 20 naturally occurring amino acids (Cornette, J.,et al, J. Mol. Biol. (1987) 195:659) are those developed on the basis ofexperimental data by Kyte and Doolittle (Kyte, J. and R. F. Doolittle,J. Mol. Biol. (1982) 157:105), and by Fauchere and Pliska (Fauchere, J.and V. Pliska, Eur. J. Med. Chem. (1983) 18:369). The Kyte/Doolittlescale measures the H₂O/organic solvent partition of individual aminoacids. Because it considers the position of amino acids in foldedproteins, it may most accurately reflect native hydrophobicity in thecontext of proteins. The Fauchere/Pliska scale measures the octanol/H₂Opartitioning of N-acetyl amino acid amides, and most accurately reflectshydrophobicity in the context of denatured proteins and/or smallsynthetic peptides. To obtain scores for hydrophobicity, each amino acidresidue was ranked on both the Kyte/Doolittle and Fauchere/Pliskahydrophobicity scales. An average rank between the two scales wascalculated and the average difference in hydrophobicity for each pairwas calculated.

Finally, for calculating amino acid side-chain volume, the partialvolume in solution obtained by noting the increase in volume of waterafter adding either one molecule or one gram of amino acid residue wasconsidered (Zamyatnin, A. A., Ann. Rev. Biophys. Bioeng. (1984) 13:145;Zamyatnin, A. A., Prog. Biophys. Mol. Biol. (1972) 24:107). The absolutedifference in the partial volume of each possible pairing of the 20naturally occurring amino acids was calculated and ranked, where Iindicated residues with the most similar volumes, and 20 the mostdissimilar.

Preparation C Materials for Assays

1. APC Lines

Cell lines that present peptides in the context of HLA-A2.1 wereprepared as follows:

The 0.221A2.1 cell line was generated by transfecting the HLA-A2.1 geneinto the HLA-A, -B, -C-null mutant EBV-transformed humanB-lymphoblastoid cell line 3A4-721.221 (Kawashima, I., et al., HumanImmunol. (1998) 59:1). The cell line GM3107 was used as APCs to measureB7 CTL responses.

Tumor cell lines were prepared by transfection of Meth A cells, amethylcholanthrene-induced sarcoma, and the Jurkat cell line with theHLA-A2.1 or HLA-A2.1/K^(b) transgene transfection was performed usingmethods described elsewhere (Vitiello, A., et al., J. Exp. Med. (1991)173:1007). A combination of the HLA-typed melanoma cell lines 624mel(A2.1⁺, MAGE⁺) and 888mel (A2.1⁻, MAGE⁻), were kindly provided by Y.Kawakami and S. Rosenberg (National Cancer Institute), and were used tomeasure presentation of endogenously processed MAGE3 epitopes (Boon, T.,et al., Ann. Rev. Immunol. (1994) 12:337). The melanoma cell lines weretreated with 100 IU/ml human IFNγ (Genzyme, Cambridge, Mass.) for 48 hat 37° C. before using as APC.

All cells in this study were grown in RPMI-1640 medium supplemented withantibiotics, sodium pyruvate, nonessential amino acids, and 10% (v/v)heat-inactivated FBS.

2. In Vitro Induction of CTL from Human PBMC and Derivation of Human CTLLines

To generate peptide-specific CTL lines against the MAGE3.112, MAGE2.170,and a carcinoembryonic antigen (CEA) epitope, CEA.691, PBMC from normalsubjects were stimulated repeatedly in vitro with peptide as described(Kawashima, I., et al., Human Immunol. (1998) 59:1). Briefly,peptide-pulsed dendritic cells (differentiated from adherent PBMC byculturing in GM-CSF and IL4) were co-cultured with autologous CD8⁺ Tcells, obtained by positive selection with antibody-coated beads (DynalA. S., Oslo, Norway or Miltenyi Biotec, Auburn, Calif.) in a 48-wellplate. After 7 days of culture in the presence of IL2, IL7, and IL10,each PBMC culture (well) was restimulated in vitro with adherent PBMCpulsed with peptide. Cultures were then tested for CTL activity bymeasuring IFNγ production after stimulation with 0.221A2.1 tumor APC (A2epitopes) or GM3107 tumor cells (B7 epitopes), in the presence orabsence of peptide. CTL lines were expanded from PBMC culturesdemonstrating peptide-specific IFNγ responses by additional in vitrostimulation with adherent peptide-pulsed PBMC.

3. Murine CTL Lines

CTL lines against epitopes HBV Pol.455 and HIV Pol.476 peptides weregenerated in HLA-A2.1/K^(bxs) transgenic mice by DNA immunization asdescribed elsewhere (Ishioka, G., et al., J. Immunol. (1999) 162:3915).HLA-A2.1/K^(bxs) and HLA-A2.1/K^(bxd) transgenic mice were bred atEpimmune. These strains represent the F1 generation of a cross betweenan HLA-A2.1/K^(b) transgenic strain generated on the C57BL/6 background(Vitiello, A., et al., J. Exp. Med. (1991) 173:1007), and SJL or BALB/cmice (Jackson Laboratories, Bar Harbor, Me.), respectively. A CTL lineagainst the MAGE2.157 epitope was generated by immunizing 8-12 wk oldHLA-A2.1/K^(bxs) mice s.c. at the tail base with 50 μg of peptide and140 μg of the HBV Core.128 Th epitope, TPPAYRPPNAPIL (SEQ ID NO:30),emulsified in IFA and restimulating primed splenocytes repeatedly invitro with peptide.

Preparation D Assay Methods

1. Measurement of Peptide Binding Affinity for HLA-A2.1 or HLA-B7Molecules

Binding of test peptides to HLA-A2.1 was measured by determining thelevel of competition induced by a given test peptide for binding of aradiolabeled standard peptide to HLA-A2.1. The percentage of MHC-boundradioactivity was determined by gel filtration and the concentration oftest peptide that inhibited 50% of the binding of the labeled standardpeptide (IC₅₀) was calculated (Ruppert, J., et al, Cell (1993) 74:929;Sette, A., et al, Mol. Immunol. (1994) 31:813). The standard peptide wasthe HBV Core. 18 epitope (sequence FLPSDFFPSV) (SEQ ID NO:31). A similarassay was performed to determine the binding affinity of peptides topurified HLA-B7 (B*0702) molecules. In the latter assay, theradiolabeled standard peptide was the SS 5-13a (L₇→Y) peptide (sequenceAPRTLVYLL) (SEQ ID NO:39).

2. Measurement of Murine and Human IFNγ, IL5, and IL10 Production by CTL

An in situ capture ELISA was used for measuring IFNγ release from CTL(McKinney, D., et al., J. Immunol. Methods (2000) 237:105). Briefly, CTLwere stimulated with APC and peptide in ELISA-grade 96-well flat bottomwells that were precoated with either an anti-mouse IFNγ (clone R4-6A2,Pharmingen, San Diego, Calif.) or anti-human IFNγ mAb (clone NIB42,Pharmingen). After culturing cells, wells are washed and developed byadding a biotinylated anti-mouse IFNγ (clone XMG1.2, Pharmingen) oranti-human IFNγ (clone 4S.B3, Pharmingen) mAb followed byenzyme-conjugated streptavidin (Zymed, South San Francisco, Calif.) and3, 3′, 5, 5′ tetramethylbenzidine substrate (ImmunoPure TMB substratekit, Pierce, Rockford, Ill.). The absorbance of each well was measuredat 450 nM on a Labsystems Multiskan RC ELISA plate reader. The level ofIFNγ produced in each well was determined by extrapolation from a mouseor human IFNγ standard curve established in the same assay.

Murine and human IL5 and IL10 were measured in culture supernates usingELISA kits (R&D Biosystems, Minneapolis, Minn.). These assays, employingthe quantitative sandwich ELISA technique, were performed according tothe manufacturer's protocol.

3. Enzyme-Liked Immunospot (Elispot) Assay for Measuring Ex Vivo CTLResponses

Elispot assays were performed according to standard protocols(Murali-Krishna, K., et al., Immunity (1998) 8:177; Lewis, J. J., etal., Int. J. Cancer (2000) 87:391). Briefly, flat bottom 96-wellnitrocellulose plates (Immobilon-P membrane, Millipore, Bedford, Mass.)were coated with anti-IFNγ mAb (10 μg/ml, clone R4-6A2) and incubatedovernight at 4° C. After washing with PBS, plates were blocked with RPMImedium containing 10% FBS for 1 h at 37° C. Four×10⁵ splenic CD8⁺ cellsisolated by magnetic beads (Miltenyi, Auburn, Calif.) and 5×10⁴Jurkat-A2.1/K^(b) cells pulsed with 10 μg/ml of peptide were added toeach well and cells were incubated for 20 h in RPMI medium containing10% FBS. After incubation, the plates were washed thoroughly withPBS/0.05% Tween and biotinylated anti-IFNγ mAb (2 μg/ml, clone XMG1.2)was added to each well and plates were incubated for 4 h at 37° C.Plates were then washed four times with PBS (containing 0.1% Tween-20)and Vectastain ABC peroxidase (Vectastain Elite kit; VectorLaboratories, Burlingame, Calif.). After incubating for 1 h at roomtemperature, plates were washed three times with 1×PBS/0.05% Tweenfollowed by three additional washes with 1×PBS. One hundred μl of AECsolution (Sigma Chemical, St. Louis, Mo.) was added to develop thespots. The reaction was stopped after 4-6 min under running tap water.The spots were counted by computer-assisted image analysis (Zeiss KSElispot Reader, Jena, Germany). The net number of spots/10⁶ CD8⁺ cellswas calculated as follows: [(number of spots against relevantpeptide)−(number of spots against irrelevant control peptide)]×2.5.

Example I Screening of Peptide Analogs for Heteroclitic Activity

A. Identification of CEA.691 and MAGE3.112 Analogs Associated withIncreased IFNγ Release

Prior to screening analogs, a peptide dose titration of IFNγ productionfrom CTL lines was performed over a wide range of doses of wildtypepeptide. 0.221A2.1 tumor cells were pulsed with varying doses of peptidethen 10⁵ peptide-loaded cells were cultured with an equivalent number ofmurine or human CTL. After 24 hr (murine) or 48 hr (human) incubation at37° C., levels of IFNγ released by CTL were measured by the in situcapture ELISA assay. After determining a dose titration curve, asuboptimal peptide dose where activity against wildtype peptide wasbarely detectable was selected for screening the antigenicity of a panelof peptide analogs. For all of the murine and human CTL lines, thissuboptimal dose ranged from 0.1-1 μg/ml. It should be noted thatalthough murine CTL lines were generated in HLA-A2.1/K^(bxs) transgenicmice which express an HLA molecule with murine H-2 K^(b) sequences inthe third domain, all responded to peptide presented on APC expressingthe native HLA-A2.1 molecule.

For screening of peptide analogs, 0.221A2.1 cells were pulsed with eachanalog at the selected suboptimal dose and peptide-loaded APC werecultured with CTL as described above. Analogs inducing enhanced CTLresponses relative to wildtype peptide were then selected for furthercharacterization. These analogs were characterized by performing apeptide dose titration side-by-side with the wildtype epitope underidentical conditions described above.

CTL lines specific for the HLA-A2.1-restricted CEA.691 and MAGE3.112epitopes were derived by repeated in vitro restimulations of human PBMCswith peptide-loaded dendritic cells or adherent monocytes, as describedin Preparation C.

A total of 117 CEA.691 and 116 MAGE3.112 analogs were generated bysystematically replacing each residue with 17 different single aminoacids. CEA.691 is IMIGVLVGV (SEQ ID NO: 1); MAGE3.112 is KVAELVHFL (SEQ.ID. NO: 4). The residues Cys, Trp and Met were in general avoided unlessthey corresponded to conservative changes. Substitutions were introducedat all positions in the peptide except at the main MHC anchor positions,position 2 and the C-terminus.

These analogs were then tested in vitro for their antigenicity. Asdescribed above, preliminary dose titration experiments for each CTLline were performed to define an antigen concentration at which IFNγproduction in response to wildtype peptide was barely detectable. Thissuboptimal concentration was then used subsequently for all antigenicityanalysis on analog peptides for each epitope, to identify analogsassociated with increased T cell stimulatory capacity. Results of suchantigenicity analysis are shown in FIG. 1. As shown in FIG. 1A, thesuboptimal 100 ng/ml dose the wildtype CEA.691 peptide yielded onlymarginal IFNγ production (<50 pg/well). By contrast, at the same dose,several CEA.691 analogs (M3, L4, P4, H5, L5, H6, T6, and 17) induceddetectable levels of IFNγ production, in the 150 to 350 pg/well range.As shown in FIG. 1B, MAGE3.112-specific CTL line 100 ng/ml of wildtypepeptide induced the release of 100 pg/ml of IFNγ, whereas two analogs(I5 and W7) were associated with inducing IFNγ levels of over 300pg/well.

All analogs of CEA.691 and MAGE3.112 that stimulated IFNγ above 100pg/well were chosen for further characterization and a complete dosetitration was carried out to identify heteroclitic analogs. Heterocliticanalogs are those that stimulate significant IFNγ release (>100 pg/well)at 10-fold or lower peptide concentrations than wildtype peptide. Forthe CEA.691 epitope two different analogs, M3 (SEQ ID NO: 2) and H5 (SEQID NO: 3), were identified. As seen in FIG. 1C, for epitope CEA.691, thewildtype peptide yielded a significant detectable IFNγ signal in the 1to 100 μg/ml dose range, while the analogs M3 and H5 stimulatedsignificant release with as little as 0.01 ng/ml of peptide. By thesecriteria, these two CEA.691 analogs are, on a molar basis, 100,000-foldmore potent in terms of IFNγ release than their unmodified wildtypecounterpart.

Similarly, for the MAGE3.112 epitope two heteroclitic analogs, I5 andW7, were identified. As shown in FIG. 1D, 1 μg/ml of wildtype peptideconcentration is required for significant IFNγ release whereas 0.1 ng/mlof either I5 (SEQ ID NO:5) or W7 (SEQ ID NO:6) analogs was required tostimulate an equivalent response. This corresponds to a greater than100,000-fold increase in biological activity compared to wildtypepeptide.

In general, the modification of a wildtype Class I epitope bysubstitution with a conservative or semi-conservative amino acid atposition 3 and/or 5 and/or 7 of the epitope to generate a heterocliticanalog enhances the immune response to the corresponding wildtypeepitope. The heteroclitic analogs not only induced a dose responseshift, but also stimulated CTL's to produce higher levels of IFNγcompared to wildtype peptide so that the maximal dose response (plateau)reached in response to the analog was much higher than the responseobtained in response to the unmodified antigen.

Example 2 Identification of Additional Heteroclitic Analogs

Three additional A2.1-restricted epitopes, the MAGE2.157 YLQLVFGIEV, SEQID NO: 7 tumor epitope, and two epitopes from viral antigens, HBVPol.455, GLSRYVARL (SEQ ID NO: 16) and HIV Pol.476 ILKEPVHGV (SEQ ID NO:18), were analyzed. All of these epitopes have previously been shown tobe immunogenic for CTL.

A panel of 240 different analogs was synthesized which included fiveconservative and five non-conservative amino acid substitutions atepitope positions 3, 5, 7 in each of the three epitopes, as well as atepitope positions 1, 4, 6, using the amino acid conservancy assignmentsdescribed in the Preparation B and in Table 2. These analogs were testedfor heteroclicity using murine CTL lines generated in HLA-A2.1/K^(bxs)transgenic mice and following an experimental strategy similar to theone described in Example 1 for the CEA.691 and MAGE3.112 epitopes.Murine CTL lines derived from HLA transgenic mice were used instead ofhuman CTL lines due to technical ease associated with generating andmaintaining mouse lines.

The results are shown in FIG. 2A (MAGE2.157), 2B (HBV Pol.455), and 2C(HIV Pol.476) with a corresponding dose titration profile for HIVPol.476 in FIG. 2D. (See Example 3 for MAGE2.157 and HIV Pol.455).

Analysis of a total of 85 different analogs of the MAGE2.157 epitopetested resulted in identification of two heteroclitic analogs, I5 (SEQID NO: 8) and F5 (SEQ ID NO: 9), that stimulated IFNγ responses at 100-to 100,000-fold lower doses than wildtype peptide (Table 1); both ofthese analogs had substitutions that were conservative orsemi-conservative in nature occurring at an odd-numbered position in thecenter of the peptide (position 5).

For the HIV Pol.476 epitope, out of 78 different analogs screened, twowere identified as having heteroclitic activity (H3 (SEQ ID NO: 19) andL3 (SEQ ID NO: 20)) (Table 1); both analogs carried either aconservative or semi-conservative substitution at an odd-numberedposition in the center of the peptide. one heteroclitic analog of HIVPol.455 epitope out of 77 tested was identified; this analog had aconservative substitution (P) at position 7 of the peptide (SEQ ID NO:17) (Table 1). An additional HIV Pol.476 analog is prepared and tested(ILIEPVHGV) (SEQ ID NO: 53).

Thus, data obtained from 240 analogs for three additional epitopes oftumor and viral origin (MAGE2.157, HIV Pol.476, and HBV Pol.455), wereconsistent with the analysis of the MAGE3.112 and CEA.691 epitopes asset forth in Example 1.

Heteroclicity analysis was also performed on two p53 epitopes. Oneepitope, p53.149M2, SMPPPGTRV (SEQ ID NO: 10) represents a fixed anchoranalog of a human p53 epitope having a methionine residue substitutionwhich enhances MHC binding. The second epitope, p53 Mu.184, GLAPPQHLIRV(SEQ ID NO: 13) has a sequence that is completely conserved between miceand humans (Theobald, et al., 92 (26): 11993 (1995)).

Dose titration analysis performed on the p53.149M2 revealed optimal andsuboptimal responses at 1 μg/ml and 0.1 μg/ml dose range. A panel of 76analogs for p53.149M2 (five conservative and five non-conservativesubstitutions at each position) was screened and only two analogs, C1(SEQ ID NO: 11) and P7 (SEQ ID NO: 12), were identified both giving IFNγrelease of 100 pg/well at a suboptimal dose, FIG. 5. On furtheranalysis, both analogs induced significant IFNγ production at 10-foldlower concentrations than wildtype peptide. In addition, the C1 analogalso induced significant IL10 levels at 100-fold lower peptideconcentrations, FIG. 6.

For the p53mu.184 epitope optimal and suboptimal levels of peptide weredetermined to be 500 ng/ml and 10 ng/ml respectively after performing adose titration analysis. A panel of 63 conservative andsemi-conservative substitution analogs were tested for immunogenicity.Two analogs with enhanced immunogenicity were found—T3 (SEQ ID NO: 14)and T3,E6 (SEQ ID NO: 15). See FIGS. 7 and 8.

Example 3 Lymphokine Profile Induced by Heteroclitic Analogs

Heteroclitic analogs have been shown previously to differentiallyactivate cytokine production from T cells whereby some analogsspecifically activate T cells to produce Th1 cytokines whereas otherspreferentially activate the production of Th2 cytokines. To investigatethe pattern of lymphokine release associated with the heterocliticanalogs of the invention, the production of Th2 cytokines IL5 and/orIL10 from CTL lines was compared to the production of IFNγ.Representative data from two different epitopes are shown in FIGS. 3 and4.

FIGS. 3A and 3B show the lymphokine profile induced by MAGE2.157analogs. IFNγ (A) and IL10 (B) produced by MAGE2.157-specific CTL's inresponse to 0.221A2.1 targets pulsed with analogs I5 or F5, or wildtype(WT) peptide was measured over several different doses. Dotted linesindicate significant levels of IFNγ (100 pg/well) or IL10 (50 pg/ml). Asseen in FIG. 3A, the F5 and I5 analogs of MAGE2.157 induced significantlevels of IFNγ production at 100-fold or 10,000-fold lowerconcentrations than wildtype peptide respectively. Moreover, the sameanalogs also induced significant IL10 production at 10-fold or 100-foldlower peptide concentrations than wildtype peptide.

Data from another epitope, HBV Pol.455, depicting the same trend areshown in FIGS. 4A and 4B. IFNγ (A) or IL10 (B) released by HBV Pol.455CTL's in response to analog P7 or wildtype (WT) peptide over severaldifferent peptide doses are shown. Once again, the P7 analog of HBVPol.455 induced significant levels of IFNγ (FIG. 4A) and IL10 (FIG. 4B)at 100-fold lower peptide concentrations than wildtype peptide. Takentogether the data summarizing all the heteroclitic analogs tested forinduction of Th2 cytokines (Table 6) indicates that most heterocliticanalogs stimulate increased production of both of Th1 and Th2 cytokines.

Example 4 HLA-A2.1 Binding Affinity of Heteroclitic Analogs

To verify that the enhanced recognition by CTL lines observed was notdue to a fortuitous increase in MHC binding capacity of the analogepitope, the MHC binding affinity of all heteroclitic analogs wasmeasured in vitro utilizing purified HLA-A2.1 molecules, and compared totheir unmodified wildtype counterparts as described in Preparation D.

As summarized in Table 6, three analogs (MAGE3.112 W7, HIV Pol.476H3,and HIV Pol.476 L3) bound to HLA-A2.1 with four-fold or higher affinitythan wildtype peptide and two analogs bound with lower affinity(MAGE2.157 I5, MAGE2.157 F5). The four remaining heteroclitic analogs,MAGE3.112 I5, CEA.691 M3, CEA.691 H5, and HBV Pol.455 P7, wereassociated with little or no change in HLA-A2.1 binding capacity.Collectively these data suggest a lack of correlation between increasedbinding and heteroclicity.

Example 5 Prediction and Immunogenicity of Analogs for the Murinep53.261 Epitope

To test for immunogenicity in vivo, the HLA-A2.1-restricted murinep53.261 epitope was used since CTL responses against this epitope havebeen shown to be partially tolerized in HLA-A2.1/K^(b) transgenic mice.This permits analysis of the capacity of predicted heteroclitic analogsto break T cell tolerance in vivo. Although heteroclitic analogsheretofore have been detected through in vitro screening with CTL linesraised against wildtype epitopes, we reasoned that analogs identified bythe substitution rules could potentially induce CTL in vivo that wereheteroclitic against the wildtype epitope, an application of interestfor designing vaccines against tolerant tumor-associated epitopes.

Immunogenicity for the p53.261 predicted analogs were tested inHLA-A2.1/K^(bxd) transgenic mice by co-immunizing mice with 50 μg of thep53.261 epitope (LLGRDSFEV) (SEQ ID NO:21) or its predicted analogs and140 μg of HBV Core. 128 helper epitope in IFA. Eleven days later, primedspleen cells were harvested and cultured in vitro with irradiatedsyngeneic LPS-activated spleen cells that had been pulsed with 10 μg/mlof peptide. After 10 days of culture, CTL were restimulated withpeptide-pulsed LPS blasts in the presence of Con A-conditioned media asa source of IL2 (Ishioka, G., et al., J. Immunol. (1999) 162:3915).Spleen cells from mice immunized with the predicted analogs werestimulated in vitro against both wildtype peptide (to determine thecross-reactivity, avidity and precursor frequency of CTL's that respondto wildtype antigen) and the respective immunizing analog (to determineavidity and precursor frequency of CTL's responding to the analog). Allshort-term, bulk populations of CTL were tested for peptide specificityby the IFNγ in situ ELISA assay 5 days after the second restimulation invitro, using Jurkat-A2.1 tumor cells as APC. Alternatively, CTLresponses were performed on freshly isolated spleen cells from immunizedanimals using the Elispot assay.

A panel of nine analogs of the p53.261 epitope consisting of threeconservative or semi-conservative substitutions at positions 3, 5, and 7of the 9-mer peptide was tested for immunogenicity in HLA-A2.1/K^(bxd)transgenic mice. Immunization of mice with each of the nine analogs andin vitro expansion of primed splenocytes with the respective immunizinganalog resulted in identification of six analogs (L7, D3, H7, H3, N5,G5) that gave CTL responses characterized by IFNγ production of 100pg/well at much lower peptide concentrations compared to CTL induced invivo and expanded in vitro with wildtype peptide.

Spleen cells from mice immunized with either WT peptide or the indicatedanalogs were stimulated in vitro with the corresponding immunizingpeptide (FIGS. 9A, B) or with WT peptide (FIGS. 9C, D). IFNγ release bythese CTL's was then measured over a dose range against targets pulsedwith the immunizing peptide (FIGS. 9A, B) or with WT peptide (FIGS. 9C,D). IFNγ release at 100 pg/well is shown as a dotted line. These resultsindicate that a significant percentage of the analogs induce CTL of ahigher avidity than those induced by wildtype peptide itself.

The cross-reactivity of CTL primed with these heteroclitic analogsagainst wildtype peptide is shown in FIG. 9C and FIG. 9D. While CTL'sobtained from animals immunized and restimulated with wildtype peptideinduced 100 pg/well IFNγ at peptide doses between 0.1-110 μg/ml, CTL'sobtained from animals immunized with analogs L7, H3, and D3, andstimulated and tested in vitro with wildtype peptide, required 10-,100-, or 1000-fold lower doses of wildtype peptide respectively, toinduce 100 pg/well of IFNγ (FIG. 9C). This suggests that in three out ofsix cases the predicted heteroclitic analogs were 10-1000-fold moreactive/potent at inducing CTL's reactive to wildtype peptide insituations where partial CTL tolerance to wildtype antigen exists.

Example 6 Cross Reactivity with Wildtype

The cross-reactivity of CTL induced by the D3 and H3 analogs were alsotested against the wildtype epitope naturally processed by ap53-expressing Meth A tumor cell clone transfected with HLA-A2.1/K^(b);it was found that CTL generated by p53.261 analogs that are heterocliticfor wildtype epitope respond to endogenously-processed p53.261 epitopepresented by Meth A/A2.1K^(b) tumor cells.

The CTL population (10⁵/well) were cultured with 2.5×10⁴ Meth A tumorcells or with a Meth A clone transfected with HLA-A2.1/K^(b) and IFNγrelease was measured by the in situ ELISA assay. As shown in FIG. 10,CTL lines raised against both D3 and H3 analogs of the p53.261 epitoperesponded to the endogenous epitope expressed by a Meth A/A2.1K^(b)tumor cell clone but not to the parental HLA-A2.1-negative Meth A tumorcell line.

Example 7 Precursor Frequency Analysis Using Elispot Assays

To confirm that cross-reactive CTL against wildtype peptide aregenerated in mice immunized with analogs CD8⁺ cells were isolated fromspleen cells of mice immunized with analogs or wildtype peptide, withoutfurther CTL expansion in vitro and the precursor frequency of CTLreactive against either wildtype or analog was determined using anElispot assay.

CD8⁺ cells isolated from mice immunized with either WT peptide or theD3, H3, L7, and H7 analogs were analyzed for their ability to releaseIFNγ when stimulated in the Elispot assay with WT peptide. FIG. 11 showsthat while the precursor frequencies of wildtype peptide-reactive CTLwere 1/66,000 (15 spots/10⁶) in mice immunized with wildtype peptide,precursor frequencies of wildtype peptide-reactive cells in miceimmunized with predicted analogs were approximately 1/15,000 for analogsD3, H3, and L7 (60-75 spots/10⁶ cells), and 1/83,000 (12 spots/10⁶) foranalog H7. This indicates wildtype-reactive cells were present at afour-fold higher frequency in mice immunized with three out of the fouranalogs compared to mice immunized with the native peptide. This findingis significant since it implies that in vivo immunization withheteroclitic analogs does indeed induce a higher number of CTL reactiveagainst wildtype peptide, using a more direct assay system where invitro expansion of in vivo-primed CTL is avoided.

Example 8 Heteroclitic Analogs Induce Human CTL Capable of RecognizingTumor Cells In Vitro

Immunogenicity of heteroclitic analogs of MAGE3.112 was also tested byinducing primary CTL from PBMC, as described in Preparation C, againsteither the MAGE3.112 peptide or the I5 and W7 analogs of this epitope.After two rounds of in vitro stimulation, PBMC cultures in 48-wells werescored positive for CTL induction if the net IFNγ production was >100pg/well and production was at least two-fold above background, afterstimulating with 0.221-A2.1 APC in the presence or absence of peptide.

To underline the physiologic relevance of our observations to humantumor antigens, we examined whether heteroclitic analogs of theMAGE3.112 epitope could induce human CTL's in a primary in vitroinduction system. Fresh naïve human PBMC from normal donors werestimulated repetitively in vitro with either wildtype or analogs asdescribed previously (Kawashima, I., et al., Human Immunol. (1998)59:1). Peptide-specific CTL responses were detected in culturesstimulated with either wildtype peptide (FIG. 12A) or the I5 (FIG. 12B)and W7 analogs (FIG. 12C). Briefly, 0.221A2.1 cells were pulsedovernight with 10 μg/ml of WT peptide (FIG. 12A), the I5 (FIG. 12B)analog, or the W7 analog (FIG. 12C). IFNγ production by CTL's growing inindividual wells from a 48-well plate were tested against 0.221A2.1cells in the presence or absence of peptide, or against the endogenousepitope-negative 888mel and the endogenous epitope-positive 624mel tumorcell lines. Only wells showing a positive peptide-specific CTL responseare shown.

More importantly cultures induced with these analogs recognized the624mel tumor cell line that endogenously processes and presents thewildtype sequence. This demonstrates that heteroclitic analogs caninduce physiologically relevant human CTL's that recognizeendogenously-generated wildtype peptide presented by tumor cells andthat the phenomenon is relevant in both human and in transgenic mousesystems.

Example 9 Synthesis and Analysis of Heteroclitic Analogs Derived fromthe HLA-A2.1 Supermotif on HLA A2 Superfamily Members

To further validate the heteroclitic substitution rules for other HLAmolecules within the A2 superfamily, the panel of nine analogs of thep53.261 epitopes discussed above consisting of threeconservative/semiconservative substitutions at positions 3, 5 and 7 aretested for in vivo immunogenicity in transgenic mice expressing one ofthe following human HLA molecules: A*0202, A*0203, A*0204, A*0205,A*0206, A*0207, A*0209, A*0214, A*6802 and A*6901.

CTLs from the mice immunized with the above-described analogs are testedfor induction of at least 100 pg/well of IFNγ production. This IFNγproduction typically occurs at much lower peptide concentrations thanthose induced and restimulated with wildtype peptide (e.g., the p53.261epitope). These results indicate that our predicted heteroclitic analogsare more potent at inducing higher avidity CTL against the nativewildtype epitope than wildtype peptide itself.

Typically, CTLs obtained from animals immunized and restimulated with awildtype peptide will induce 100 pg/well IFNγ at peptide doses of 5-10μg/ml, whereas CTLs obtained from animals immunized with theabove-described analogs, and stimulated and tested in vitro withwildtype peptide, require 10-fold, 100-fold or even 1000-fold lowerdoses of wildtype peptide respectively, to induce 100 pg/well of IFNγ.

Example 10 Identification of Heteroclitic Analogs of a B7 SuperfamilyCTL Epitope, MAGE2.170

To better define the application of the invention to HLA Supertypefamilies other than HLA-A2, analogs of the B7 superfamily epitopeMAGE2.170 (sequence VPISHLYIL) (SEQ ID NO:46) were synthesized andscreened in a fashion similar to that described previously for A2superfamily epitopes. A panel of analogs of the MAGE2.170 epitopeconsisting of conservative/semi-conservative and non-conservativesubstitutions at every non-anchor position were screened at twosuboptimal peptide doses using a human CTL line generated against thewildtype epitope. As previously described, this screening assay servedto identify any potentially heteroclitic analogs that induce strongerCTL responses compared to wildtype peptide.

As shown in FIG. 13, analogs substituted at position 7 with either a H,M, E, G, or D residue stimulated IFNγ responses that were greater thanthe wildtype peptide when tested at the 0.01 μg/ml dose. When thestimulatory capacity of these five analogs were further analyzed in apeptide dose titration using the same wildtype epitope-specific CTLline, all of them demonstrated strong heteroclitic activity inasmuch asthey all stimulated an equivalent level of IFNγ production (e.g. 200pg/well) at >10-fold lower doses compared to the wildtype epitope, andthe magnitude of response stimulated by the analogs was >2-fold greaterthan wildtype epitope at several peptide doses (FIG. 14).

To determine whether the heteroclitic activity of MAGE2.170 analogs wascorrelated with an increase or decrease in MHC binding activity, thebinding affinity of the H7, M7, E7, G7, and D7 analogs to purifiedHLA-B7 molecules was determined relative to the wildtype epitope.Results shown in Table 7 indicate that there was no correlation betweenMHC binding of the analogs and heteroclicity inasmuch as 4 of the 5MAGE2.170 analogs demonstrated binding affinities within a two-foldrange of the wildtype peptide. The fifth epitope, MAGE2.170 D7,demonstrated a >100-fold decrease in binding compared to the wildtypepeptide, therefore an enhancement in MHC binding could not account forthe heteroclitic activity observed with this analog.

In summary, these results indicate that heteroclitic analogs can begenerated from a B7 superfamily epitope by introducing single amino acidsubstitutions and that the substitution pattern showed similarity anddifferences with A2 heteroclitic epitopes. Like A2 heterocliticepitopes, heteroclitic analogs of the B7 superfamily epitope MAGE2.170could be generated by introducing substitutions at an odd-numberposition in the middle of the peptide (position 7). The nature of thesubstitutions for the MAGE2.170 epitope was eitherconservative/semi-conservative (the Y→H and Y→M substitutions) ornon-conservative (the Y→E, Y→G, and Y→D substitutions) compared to thenative residue (Table 7). Thus, the observation that non-conservativesubstitutions can result in heteroclitic analogs for the MAGE2.170 CTLepitope indicate a partially overlapping substitution pattern than thatobserved with A2 superfamily epitopes.

Example 11 Synthesis and Analysis of Heteroclitic Analogs Derived fromthe HLA-B7 Supermotif on HLA B7 Superfamily Members

To further validate the heteroclitic substitution rules, additionalstudies are carried out with heteroclitic analogs derived from a peptidebearing a sequence within the HLA-B7 supermotif. For example, theanalogs can be tested for in vivo immunogenicity.

For this study, the HLA-B7 supermotif bearing peptide, APRTLVYLL (SEQ.ID. NO:39) epitope is chosen and synthesized. A panel of analogsconsisting of three conservative/semiconservative substitutions atpositions 3, 5 and 7 of the 9-mer peptide, are tested for immunogenicityin HLA-B*0702/K^(b) transgenic mice. The panel includes APETLVYLL (SEQID NO:40), APRTWVYLL (SEQ ID NO:41), and APRTLVPLL (SEQ ID NO:42),corresponding to a semi-conservative change is the third, fifth andseventh position, respectively.

CTLs from the mice immunized with the above-described analogs are testedfor induction of at least 100 pg/well of IFNγ production. This IFNγproduction will typically occur at much lower peptide concentrationsthan those induced and restimulated with wildtype peptide (e.g.,APRTLVYLL) (SEQ ID NO:39). These results will indicate that ourpredicted heteroclitic analogs are more potent at inducing higheravidity CTL than wildtype peptide itself.

Typically, CTLs obtained from animals immunized and restimulated with awildtype peptide will induce 100 pg/well IFNγ at peptide doses of 5-10μg/ml, whereas CTLs obtained from animals immunized with theabove-described analogs, and stimulated and tested in vitro withwildtype peptide, require 10-fold, 100-fold or even 1000-fold lowerdoses of wildtype peptide respectively, to induce 100 pg/well of IFNγ.

To further validate the heteroclitic substitution rules for other HLAmolecules with the B7 superfamily, the peptides APETLVYLL (SEQ IDNO:40), APRTWVYLL (SEQ ID NO:41) and APRTLVPLL (SEQ ID NO:42) are testedfor in vivo immunogenicity in transgenic mice expressing one of thefollowing human HLA molecules: B*0702, B*0703, B*0704, B*0705, B*1508,B*3501, B*3502, B*3503, B*3503, B*3504, B*3505, B*3506, B*3507, B*3508,B*5101, B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501, B*5502,B*5601, B*5602, B*6701 and B*7801.

CTLs from the mice immunized with the above-described analogs are testedfor induction of at least 100 pg/well of IFNγ production. This IFNγproduction will typically occur at much lower peptide concentrationsthan those induced and restimulated with wildtype peptide (e.g.,APRTLVYLL) (SEQ ID NO:39). These results will indicate that ourpredicted heteroclitic analogs are more potent at inducing higheravidity CTL than wildtype peptide itself.

Typically, CTLs obtained from animals immunized and restimulated with awildtype peptide will induce 100 pg/well IFNγ at peptide doses of 5-10μg/ml, whereas CTLs obtained from animals immunized with theabove-described analogs, and stimulated and tested in vitro withwildtype peptide, require 10-fold, 100-fold or even 1000-fold lowerdoses of wildtype peptide respectively, to induce 100 pg/well of IFNγ.

Precursor Frequency Analysis Using Elispot Assays

To confirm that cross-reactive CTL against wildtype peptide aregenerated in mice immunized with analogs, CD8⁺ cells are isolated fromspleens immunized with analogs or wildtype peptide without further CTLexpansion in vitro. From this material, the precursor frequency of CTLreactive against either wildtype or analog using Elispot assay isdetermined. The precursor frequencies of wildtype peptide reactive CTLsare typically much lower than the precurser frequencies of the analogs.

Heteroclitic Analogs can Induce Human CTL Capable of RecognizingEpitopes In Vitro

Heteroclitic analogs can be analyzed for induction of CTLs in a primaryin vitro induction system. Fresh naïve human PBMC from normal donors arestimulated repetitively in vitro, with either wildtype or analogs, in 48well plates as described previously. Peptide specific CTL responses arethen detected in cultures stimulated with either a wildtype peptide or aheteroclitic analog. Cultures induced with these analogs can recognizetargets that are endogenously processed and present the wildtypesequence. This demonstrates that heteroclitic analogs can inducephysiologically relevant human CTLs that recognize endogenouslygenerated wildtype peptide expressed on cells and that the phenomenon isrelevant in both human and in transgenic mouse systems.

Example 12 Synthesis and Analysis of Heteroclitic Analogs Derived fromthe HLA-A3 Supermotif on HLA A3 Superfamily Members

To further validate the heteroclitic substitution rules, additionalstudies are carried out with heteroclitic analogs derived from a peptidebearing a sequence within the HLA-A3 supermotif. For example, theanalogs can be tested for in vivo immunogenicity.

For this study, the HLA-A3 supermotif bearing peptide, KVFPYALINK (SEQID NO:29) epitope is chosen and synthesized. A panel of analogs of SEQID NO:29 consisting of three conservative/semiconservative substitutionsat positions 3, 5 and 7 of the 9-mer peptide, are tested forimmunogenicity in HLA-A*3101/K^(b) transgenic mice. The panel includesKVHPYALINK (SEQ ID NO:43), KVFPQALINK (SEQ. ID. NO:44) and KVFPYAKINK(SEQ ID NO:45), corresponding to a semi-conservative change in thethird, fifth and seventh position, respectively.

CTLs from the mice immunized with the above-described analogs are testedfor induction of at least 100 pg/well of IFNγ production. This IFNγproduction typically occurs at much lower peptide concentrations thanthose induced and restimulated with wildtype peptide (e.g., KVFPYALINK)(SEQ ID NO:29). These results indicate that our predicted heterocliticanalogs are more potent at inducing higher avidity CTL against wildtypethan wildtype peptide itself.

Typically, CTLs obtained from animals immunized and restimulated with awildtype peptide induce 100 pg/well IFNγ at peptide doses of 5-10 μg/ml,whereas CTLs obtained from animals immunized with the above-describedanalogs, and stimulated and tested in vitro with wildtype peptide,require 10-fold, 100-fold or even 1000-fold lower doses of wildtypepeptide respectively, to induce 100 pg/well of IFNγ.

To further validate the heteroclitic substitution rules for other HLAmolecules with the A3 superfamily, the peptides KVHPYALINK (SEQ IDNO:43), KVFPQALI NK (SEQ ID NO:44) and KVFPYAKINK (SEQ ID NO:45) aretested for in vivo immunogenicity in transgenic mice expressing one ofthe following human HLA molecules: A*0301, A*1101, A*3101, A*3301 andA*6801.

CTLs from the mice immunized with the above-described analogs are testedfor induction of at least 100 pg/well of IFNγ production. This IFNγproduction typically occurs at much lower peptide concentrations thanthose induced and restimulated with wildtype peptide (e.g., KVFPYALINK)(SEQ ID NO:29). These results will indicate that our predictedheteroclitic analogs are more potent at inducing higher avidity CTL thanwildtype peptide itself.

Typically, CTLs obtained from animals immunized and restimulated with awildtype peptide induce 100 pg/well IFNγ at peptide doses of 5-10 μg/ml,whereas CTLs obtained from animals immunized with the above-describedanalogs, and stimulated and tested in vitro with wildtype peptide,require 10-fold, 100-fold or even 1000-fold lower doses of wildtypepeptide respectively, to induce 100 pg/well of IFNγ.

Precursor Frequency Analysis Using Elispot Assays

To confirm that cross-reactive CTL against wildtype peptide aregenerated in mice immunized with analogs, CD8⁺ cells are isolated fromspleens immunized with analogs or wildtype peptide without further CTLexpansion in vitro. From this material, the precursor frequency of CTLreactive against either wildtype or analog using Elispot assay isdetermined. The precursor frequencies of wildtype peptide reactive CTLsare typically much lower than the precurser frequencies of the analogs.

Heteroclitic Analogs can Induce Human CTL Capable of RecognizingEpitopes In Vitro

Heteroclitic analogs are analyzed for induction of CTLs in a primary invitro induction system. Fresh naïve human PBMC from normal donors arestimulated repetitively in vitro, with either wildtype or analogs, in 48well plates as described previously. Peptide specific CTL responses arethen detected in cultures stimulated with either a wildtype peptide or aheteroclitic analog. Cultures induced with these analogs recognizetargets that are endogenously processed and present the wildtypesequence. This demonstrates that heteroclitic analogs inducephysiologically relevant human CTLs that recognize endogenouslygenerated wildtype peptide expressed on cells and that the phenomenon isrelevant in both human and in transgenic mouse systems.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

TABLE 1 SEQ. ID NO:1 IMIGVLVGV CEA.691 SEQ. ID NO:2 IMMGVLVGV CEA.691 M3SEQ. ID NO:3 IMIGHLVGV CEA.691 H5 SEQ ID NO:4 KVAELVHFL MAGE3.112 SEQ.ID NO:5 KVAEIVHFL MAGE3.112 I5 SEQ. ID NO:6 KVAELVWFL MAGE3.112 W7 SEQ.ID NO:7 YLQLVFGIEV MAGE2.157 SEQ. ID NO:8 YLQLIFGIEV MAGE2.157 I5 SEQ.ID NO:9 YLQLFFGIEV MAGE2.157 F5 SEQ. ID NO:10 SMPPPGTRV p53.149M2 SEQ.ID NO:11 CMPPPGTRV p53.149M2 C1 SEQ. ID NO:12 SMPPPGPRV p53.149M2 P7SEQ. ID NO:13 GLAPPQHLIRV p53.Mu.184 SEQ. ID NO:14 GLTPPQHLIRVp53.Mu.184 T3 SEQ. ID NO:15 GLTPPEHLIRV p53.Mu.184 T3, E6 SEQ. ID NO:16GLSRYVARL HBV Po1.455 SEQ. ID NO:17 GLSRYVPRL HBV Po1.455 P7 SEQ. IDNO:18 ILKEPVHGV HIV Po1.476 SEQ. ID NO:19 ILHEPVHGV HIV Po1.476 H3 SEQ.ID NO:20 ILLEPVHGV HIV Po1.476 L3 SEQ. ID NO:21 LLGRDSFEV p53.261 SEQ.ID NO:22 LLDRDSFEV p53.261 D3 SEQ. ID NO:23 LLHRDSFEV p53.261 H3 SEQ. IDNO:24 LLGRDSLEV p53.261 L7 SEQ. ID NO:25 LLGRDSHEV p53.261 H7 SEQ. IDNO:26 LLGRNSFEV p53.261 N5 SEQ. ID NO:27 LLGRGSFEV p53.261 G5 SEQ. IDNO:28 APAAAAAAY SEQ. ID NO:29 KVFPYALINK A3 wildtype SEQ. ID NO:30TPPAYRPPNAPIL HBVCore.128 Th SEQ. ID NO:31 FLPSDFFPSV HBVCore.18 SEQ. IDNO:39 APRTLVYLL HLA-B7 SEQ. ID NO:48 VPISHLYIL MAGE2.170 SEQ. ID NO:49VPISHLHIL MAGE2.170 H7 SEQ. ID NO:50 VPISHLMIL MAGE2.170 M7 SEQ. IDNO:51 VPISHLGIL MAGE2.170 G7 SEQ. ID NO:52 VPISHLEIL MAGE2.170 E7 SEQ.ID NO:53 VPISHLDIL MAGE2.170 D7

TABLE 2 Compiled rankings and similarity assignments.

TABLE 3 POSITION POSITION POSITION 2 (Primary 3 (Primary C Terminus(Primary Anchor) Anchor) Anchor) SUPER- MOTIFS A1 T, I, L, V, M, S F, W,Y A2 L, I, V, M, A, T, I, V, M, A, T, L Q A3 V, S, M, A, T, L, R, K IA24 Y, F, W, I, V, L, F, I, Y, W, L, M M, T B7 P V, I, L, F, M, W, Y, AB27 R, H, K F, Y, L, W, M, I, V, A B44 E, D F, W, L, I, M, V, A B58 A,T, S F, W, Y, L, I, V, M, A B62 Q, L, I, V, M, P F, W, Y, M, I, V, L, AMOTIFS A1 T, S, M Y A1 D, E, A, S Y A2.1 L, M, V, Q, I, A, V, L, I, M,A, T T A3 L, M, V, I, S, A, K, Y, R, H, F, A T, F, C, G, D A11 V, T, M,L, I, S, K, R, Y, H A, G, N, C, D, F A24 Y, F, W, M F, L, I, W A*3101 M,V, T, A, L, I, R, K S A*3301 M, V, A, L, F, I, R, K S, T A*6801 A, V, T,M, S, L, R, K I B*0702 P L, M, F, W, Y, A, I, V B*3501 P L, M, F, W, Y,I, V, A B51 P L, I, V, F, W, Y, A, M B*5301 P I, M, F, W, Y, A, L, VB*5401 P A, T, I, V, L, M, F, W, YBolded residues are preferred, italicized residues are less preferred: Apeptide is considered motif-bearing if it has primary anchors at eachprimary anchor position for a motif or supermotif as specified in theabove table.

TABLE 4 POSITION POSITION POSITION 2 (Primary 3 (Primary C Terminus(Primary Anchor) Anchor) Anchor) SUPER- MOTIFS A1 T, I, L, V, M, S F, W,Y A2 V, Q, A, T I, V, L, M, A, T A3 V, S, M, A, T, L, R, K I A24 Y, F,W, I, V, L, F, I, Y, W, L, M M, T B7 P V, I, L, F, M, W, Y, A B27 R, H,K F, Y, L, W, M, I, V, A B58 A, T, S F, W, Y, L, I, V, M, A B62 Q, L, I,V, M, P F, W, Y, M, I, V, L, A MOTIFS A1 T, S, M Y A1 D, E, A, S Y A2.1V, Q, A, T* V, L, I, M, A, T A3.2 L, M, V, I, S, A, K, Y, R, H, F, A T,F, C, G, D A11 V, T, M, L, I, S, K, R, H, Y A, G, N, C, D, F A24 Y, F, WF, L, I, W *If 2 is V, or Q, the C-term is not L

Bolded residues are preferred, italicized residues are less preferred: Apeptide is considered motif-bearing if it has primary anchors at eachprimary anchor position for a motif or supermotif as specified in theabove table.

TABLE 5 Allelle-specific HLA-supertype members HLA-supertypeVerified^(a) Predicted^(b) A1 A*0101, A*2501, A*2601, A*2602, A*3201A*0102, A*2604, A*3601, A*4301, A*8001 A2 A*0201, A*0202, A*0203,A*0204, A*0205, A*0206, A*0207, A*0208, A*0210, A*0211, A*0212, A*0213A*0209, A*0214, A*6802, A*6901 A3 A*0301, A*1101, A*3101, A*3301, A*6801A*0302, A*1102, A*2603, A*3302, A*3303, A*3401, A*3402, A*6601, A*6602,A*7401 A24 A*2301, A*2402, A*3001 A*2403, A*2404, A*3002, A*3003 B7B*0702, B*0703, B*0704, B*0705, B*1508, B*3501, B*3502, B*3503, B*1511,B*4201, B*5901 B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101,B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501, B*5502, B*5601,B*5602, B*6701, B*7801 B27 B*1401, B*1402, B*1509, B*2702, B*2703,B*2704, B*2705, B*2706, B*2701, B*2707, B*2708, B*3802, B*3903, B*3904,B*3801, B*3901, B*3902, B*7301 B*3905, B*4801, B*4802, B*1510, B*1518,B*1503 B44 B*1801, B*1802, B*3701, B*4402, B*4403, B*4404, B*4001,B*4002, B*4101, B*4501, B*4701, B*4901, B*5001 B*4006 B58 B*5701,B*5702, B*5801, B*5802, B*1516, B*1517 B62 B*1501, B*1502, B*1513,B*5201 B*1301, B*1302, B*1504, B*1505, B*1506, B*1507, B*1515, B*1520,B*1521, B*1512, B*1514, B*1510 ^(a)Verified alleles include alleleswhose specificity has been determined by pool sequencing analysis,peptide binding assays, or by analysis of the sequences of CTL epitopes.^(b)Predicted alleles are alleles whose specificity is predicted on thebasis of B and F pocket structure to overlap with the supertypespecificity.

TABLE 6 Characterization of heterocyclic analogs identified from tumorand viral antigens. SEQ Th1 Th2 A*0201 ID Heterocyclic Type of Positionof cyto- cyto- binding Antigen NO: Sequence substitution substitutionsubstitution kines^(a) kines^(b) (IC50, nM)^(d) CEA.691 1 IMIGVLVGV None(WT) None   1  10 54 CEA.691 M3 2 IMMGVLVGV I→M Conservative 3  10⁻⁵   127 CEA.691 H5 3 IMIGHLVGV V→H Semi- 5  10⁻⁷  10⁻¹ 16 conservativeMAGE3.112 4 KVAELVHFL None (WT) None   1 NS^(c) 94 MAGE3.112 I5 5KVAEIVHFL L→I Conservative 5  10⁻⁴ NS 66 MAGE3.112 W7 6 KVAELVWFL H→WSemi- 7  10⁻⁷ NS 7 conservative MAGE2.157 7 YLQLVFGIEV None (WT) None  1  10 40 MAGE2.157 I5 8 YLQLIFGIEV V→I Conservative 5  10⁻⁴  10⁻² 476MAGE2.157 F5 9 YLQLFFGIEV V→F Semi- 5  10⁻²  10⁻² 212 conservative HBVPo1.455 16 GLSRYVARL None (WT) None  10  10 83 HBV Po1.455 P7 17GLSRYVPRL A→P Conservative 7  10⁻²  10⁻² 267 HIV Po1.476 18 ILKEPVHGVNone (WT) >10 >10 369 HIV Po1.476 H3 19 ILHEPVHGV K→H Conservative 3   1  1 78 HIV Po1.476 L3 20 ILLEPVHGV K→L Semi- 3  10⁻¹   1 63 conservative^(a)Minimum peptide concentration (μg/ml) required to induce 100 pg/wellof IFNγ (Th1 cytokines) ^(b)Minimum peptide concentration (μg/ml)required to induce 50 pg/ml of IL10 or IL5 (Th2 cytokines) ^(c)NS,cytokine levels not significant (<5 pg/ml) ^(d)A relative binding changeof four-fold or more compared to wildtype peptide is consideredsignificant and is indicated in bold

TABLE 7 Summary of heteroclitic analogs of MAGE2.170 HeterocliticPosition of B*0702 binding Antigen substitution Type of substitutionSubstitution (IC50, nM) SEQ ID NO: MAGE2.170 None (WT) None 112 SEQ IDNO: 48 MAGE2.170H7 Y --> H Semi-conservative 7 75 SEQ ID NO: 49MAGE2.170M7 Y --> M Semi-conservative 7 69 SEQ ID NO: 50 MAGE2.170G7 Y--> G Non-conservative 7 105 SEQ ID NO: 51 MAGE2.170E7 Y --> ENon-conservative 7 186 SEQ ID NO: 52 MAGE2.170D7 Y --> DNon-conservative 7 1276 SEQ ID NO: 53

1-11. (canceled)
 12. A peptide comprising an analog of a Major Histocompatibility Complex (MHC) class I peptide epitope, wherein said analog has enhanced immunogenicity compared to said epitope, and wherein said peptide analog is prepared by: a) identifying a MHC class I epitope comprising a formula (A), wherein formula (A) is Rn-R2-R3-R4-R5-R6-R7- . . . Rx, Rn is the N-terminal amino acid, Rx is the C-terminal amino acid, x=8-11 such that Rx can be from the eighth to the eleventh amino acid residue from Rn, R2 or R3 and Rx are primary anchor residues of a motif or supermotif, and b) producing a polypeptide comprising an analog, said analog comprising a formula (B) identical to said formula (A) except one or more conservative or semiconservative amino acid substitutions at R3 and/or R5 and/or R7, provided said one or more substitutions is not of a primary anchor residue. 13-15. (canceled)
 16. A composition comprising at least the peptide of claim
 12. 17. The composition of claim 16, wherein the peptide contains 9-15 amino acids.
 18. The composition of claim 16, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:20.
 19. A composition of claim 16, wherein the peptide is admixed or joined to a CTL epitope.
 20. A composition of claim 16, wherein the peptide is admixed or joined to an HTL epitope.
 21. A composition of claim 20, wherein the HTL epitope is a pan-DR binding molecule.
 22. A composition of claim 16, further comprising a liposome.
 23. A composition of claim 16, wherein the epitope is coupled to a lipid.
 24. A composition of claim 16, wherein said epitope is included in a heteropolymer.
 25. A composition of claim 16, wherein the epitope is included in a homopolymer. 26-30. (canceled)
 31. The composition of claim 16, further comprising a label.
 32. The composition of claim 31, wherein the label is biotin, a fluorescent moiety, a non-mammalian sugar, a radio label or a small molecule to which a monoclonal antibody binds.
 33. The composition of claim 16 which is a vaccine containing: a unit dosage of said peptide, and a pharmaceutical excipient. 34-39. (canceled) 